CN114807270A - 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
- Publication number
- CN114807270A CN114807270A CN202210209313.2A CN202210209313A CN114807270A CN 114807270 A CN114807270 A CN 114807270A CN 202210209313 A CN202210209313 A CN 202210209313A CN 114807270 A CN114807270 A CN 114807270A
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- CN
- China
- Prior art keywords
- burdock root
- burdock
- rhizopus nigricans
- polysaccharide
- root polysaccharide
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Links
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The invention relates to a burdock root polysaccharide prepared by utilizing rhizopus nigricans fermentation and a production process and application thereof. The cattleThe weight average molecular weight of the burdock root polysaccharide is 5 multiplied by 10 4 ～1×10 6 Da, the molar content of galactose and galacturonic acid in the burdock root polysaccharide is more than 50%. The invention also provides a production process for preparing the burdock root polysaccharide by utilizing the rhizopus nigricans fermentation. According to the invention, rhizopus nigricans has vigorous reproductive capacity, rich biological enzyme systems and stronger biotransformation capacity, the burdock root polysaccharide RB3 component is obtained by separating and purifying the burdock root and burdock root water extraction residues after fermentation, and research results of in vitro tumor cell experiments and in vivo tumor-bearing mouse experiments show that the burdock root polysaccharide RB3 has a more remarkable effect of regulating immunity and resisting tumors, can be applied to the fields of health-care food and medical drugs, and provides scientific support for development and application of rhizopus nigricans and burdock resources.
Description
Technical Field
The invention relates to a 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 cause of death worldwide. According to the world cancer report data of the world health organization IARC, 457 thousands of new cancers are found in China in 2020, which account for 23.7% of the number of new cancers worldwide and 30% of cancer death cases. Among the main cancer types in China, the highest cancer types are lung cancer, breast cancer, gastric cancer, colorectal cancer, liver cancer, esophageal cancer and the like. In addition, the incidence of malignancy remains increased by about 3.9% per year, and mortality remains increased by 2.5% per year. Compared with developed countries such as the United states, Chinese cancer treatment schemes are more limited, and on the one hand, traditional chemotherapeutic drug therapy is still an important tumor treatment means. However, the traditional chemotherapy method has poor selectivity and large toxic and side effects, strongly stimulates the digestive system, is easy to cause damage to the liver and kidney functions, and even destroys the hematopoietic function of bone marrow and the immune function of human body. The biological polysaccharide has the advantages of easy acquisition, safety, effectiveness and small toxic and side effect, and can be combined with chemotherapeutic drugs, so that the biological polysaccharide is always a hot point for the innovative research and development of antitumor drugs.
Arctium Lappa L is a biennial plant resource of food and drug homology of Compositae, and is widely planted in China, east Asia, Europe, south America and other countries and regions. Besides serving as the edible value of vegetables, the burdock roots and the extract thereof also show various potentials in the aspect of medicine, including anti-inflammation and anti-oxidation, gastrointestinal function protection, glycolipid metabolism regulation, intestinal flora improvement and immunoregulation anti-tumor activity. Wherein, the burdock polysaccharide is the main active component of the burdock root and is fructo-oligosaccharide with the molecular weight of about 2100 Da. At present, the primary processed product of the burdock root and the extract thereof form an industrial scale, and the health care and medical value of the burdock is more and more noticed and welcomed by people.
Rhizopus nigricans (Rhizopus nigricans) is a filamentous fungus belonging to the genus Rhizopus of the subdivision Zygomycotina of the phylum Eumycota, is an abundant biological enzyme, and is a microorganism species commonly used in the fermentation industry due to its biocatalytic and bioconversion capabilities. Researches on metabolites of rhizopus nigricans since 2013 find that exopolysaccharides of rhizopus nigricans have prominent effects on enhancing immunity, inducing tumor apoptosis and the like.
Chinese patent document CN103859578A discloses an extraction method of burdock crude polysaccharide and application thereof in tobacco. Taking burdock eradicate substances, cleaning, slicing, putting into a homogenizer, adding water, crushing by the homogenizer, performing ultrasonic extraction for 1-2 h, extracting for 1-2 h by using water at the temperature of 80-120 ℃, filtering, concentrating filtrate, adding ethanol into concentrated solution to enable the concentration of the ethanol to reach more than 80%, uniformly mixing, standing for more than 8h at the temperature of 4 ℃, centrifuging, filtering, and precipitating to obtain the burdock root crude polysaccharide. Although the patent discloses a preparation method of burdock polysaccharide, the method only adopts a physical method to extract the polysaccharide, 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 a burdock root polysaccharide prepared by utilizing rhizopus nigricans fermentation and a production process and application thereof.
The technical scheme of the invention is as follows:
a Burdock root polysaccharide prepared by fermenting Rhizopus nigricans has a weight average molecular weight of 5 × 10 4 ～1×10 6 Da, the molar content of galactose and galacturonic acid in the burdock root polysaccharide is more than 50%.
The invention also provides a production process for preparing the burdock root polysaccharide by utilizing the rhizopus nigricans fermentation, which comprises the following steps:
(1) inoculating Rhizopus nigricans strains into a potato glucose agar culture medium, culturing at 26-30 ℃ for 4-6 days until white hyphae are fully paved and black spores are grown, selecting Rhizopus nigricans hypha blocks, transferring into the potato glucose agar culture medium, continuously culturing for 2-4 days until a large number of black spores appear, washing the Rhizopus nigricans thalli with sterile water, and collecting the spores to obtain a Rhizopus nigricans spore suspension;
(2) selecting fresh burdock roots or burdock root water-insoluble substances or burdock root water-insoluble dry powder, adding the fresh burdock roots or burdock root water-insoluble dry powder into a shake flask of a potato glucose liquid culture medium, performing wet sterilization, then inoculating rhizopus nigricans spore suspension according to the volume ratio of 0.5-5%, and then performing fermentation culture for 7-15 days under the conditions that the temperature is 25-30 ℃ and the oscillation speed is 120-200rpm, so as to obtain burdock root fermentation liquid;
(3) filtering and clarifying burdock root fermentation liquor, concentrating supernatant, precipitating with ethanol, redissolving, deproteinizing, decolorizing, dialyzing, and 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.
Preferably, in step (1), the size of the rhizopus nigricans hyphae block is 1 × 1 cm.
Preferably, in step (1), the density of Rhizopus nigricans spores in the Rhizopus nigricans spore suspension is 1 × 10 5 ～10 8 one/mL.
According to a preferred embodiment of the invention, in step (2), the Rhizopus nigricans spore suspension is added in an amount of 2% by volume of the potato dextrose liquid medium.
Preferably, in the step (2), the fresh burdock roots are cleaned, peeled and cut, and the weight-to-volume ratio of the fresh burdock roots to the potato dextrose liquid culture medium is (1-4): 10, unit is: g/mL.
Preferably, in the step (2), the burdock root water-insoluble substance is obtained by treating fresh burdock roots through a hot water extraction method, and the weight-to-volume ratio of the burdock root water-insoluble substance to the potato dextrose liquid culture medium is (1-4): 10, unit is: g/mL.
Preferably, in the step (2), the burdock root water-insoluble dry powder is obtained by drying and crushing burdock root water-insoluble at 50-70 ℃, and the weight-to-volume ratio of the burdock root water-insoluble dry powder to the potato dextrose liquid culture medium is (1-5): 100, with units of: g/mL.
Preferably, in step (3), the anion-exchange DEAE fast flow chromatography is performed by the following method: the burdock root polysaccharide RB3 prepared by rhizopus nigricans fermentation is obtained by adopting 0.02M tris-hydrochloric acid, 0.02M tris-hydrochloric acid and 0.1M sodium chloride solution, 0.02M tris-hydrochloric acid and 0.2M sodium chloride solution, 0.02M tris-hydrochloric acid and 0.3M sodium chloride solution, 0.02M tris-hydrochloric acid and 0.4M sodium chloride solution and 0.02M tris-hydrochloric acid and 0.5M sodium chloride solution to elute according to the sodium chloride concentration from small to large in sequence and collecting the eluent of 0.02M tris-hydrochloric acid and 0.3M sodium chloride solution.
The application of the burdock root polysaccharide in preparing immunoregulation antitumor drugs or foods.
According to the invention, the burdock root polysaccharide and 5-fluorouracil are preferably used together, the using dosage of the burdock root polysaccharide is 50-150 mg/kg/d, and the using dosage of the 5-fluorouracil is 15-25 mg/kg/d.
The invention has the technical characteristics and beneficial effects that:
1. the rhizopus nigricans has vigorous reproductive capacity, rich biological enzyme systems and stronger biotransformation capacity, the burdock root polysaccharide RB3 component is obtained by separating and purifying the burdock root and burdock root water extraction residues after fermentation, the burdock root polysaccharide RB3 shows excellent immunoregulation anti-tumor activity, has higher tumor inhibition rate on tumors, has NO cytotoxic effect on normal cells, can also enhance the phagocytosis activity of the macrophages, and induces the secretion of cell factors such as NO, IL-6, TNF-alpha and the like of the macrophages. The invention not only effectively utilizes the biological resources of burdock, rhizopus nigricans and the like, but also provides scientific support for the development and application of burdock roots and rhizopus nigricans in the field of medicine.
2. The invention provides a production process for preparing burdock root polysaccharide by utilizing rhizopus nigricans fermentation, and the obtained weight average molecular weight is 5 multiplied by 10 4 ～1×10 6 Da burdock root polysaccharide RB3, burdock root polysaccharide RB3 constitute and are mainly galactose and galacturonic acid, the molar content ratio of galactose and galacturonic acid in the burdock root polysaccharide is more than 50%, research results of in vitro tumor cell experiments and in vivo tumor-bearing mouse experiments show that the burdock root polysaccharide RB3 has a relatively obvious effect of adjusting immunity and resisting tumor, can be applied to the fields of health food and medical drugs, and provide scientific support for development and application of Rhizopus nigricans and burdock resources.
Description of the drawings:
FIG. 1 is a diagram of a burdock root polysaccharide extracted from burdock root water-insoluble substances without rhizopus nigricans fermentation (left) and an agarose gel DEAE fast flow diagram of burdock root polysaccharide RB3 prepared by rhizopus nigricans fermentation of the invention (right).
FIG. 2 is an HPLC chart of the preparation of Burdock root polysaccharide RB3 by using Rhizopus nigricans fermentation.
FIG. 3 is an infrared spectrum of Burdock root polysaccharide RB3 prepared by Rhizopus nigricans fermentation.
FIG. 4 is a diagram of the analysis of polysaccharide methylated glycitol acetyl ester (PMAA) of the Burdock root polysaccharide RB3 prepared by Rhizopus nigricans fermentation.
FIG. 5 is a nuclear magnetic spectrum of Burdock root polysaccharide RB3 prepared by Rhizopus nigricans fermentation.
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 a COSY map.
FIG. 6 is a graph showing the cytotoxicity test results of Burdock root polysaccharide RB3 prepared by Rhizopus nigricans fermentation on human normal hepatocyte LO 2.
FIG. 7 is a graph showing the experimental results of the effect of fermentation of rhizopus nigricans for preparing Burdock root polysaccharide RB3 on the proliferation of mouse macrophage RAW 264.7.
FIG. 8 is a graph showing the effect of fermentation of rhizopus nigricans to prepare Burdock root polysaccharide RB3 on cytokine secretion of mouse macrophage RAW 264.7.
Detailed Description
The present invention is further described below in conjunction with examples which will enable those skilled in the art to more effectively understand the present invention, but which are not intended to limit the invention in any way.
The potato dextrose media formulations used in the following examples: 200g of potatoes, 15-20 g of cane sugar, 20-30 g of agar and 1000mL of distilled water.
Example 1
A production process for preparing burdock root polysaccharide by utilizing rhizopus nigricans fermentation comprises the following steps:
(1) inoculating Rhizopus nigricans into a plate containing potato glucose agar (PDA), culturing at 28 deg.C for 5 days until white hypha is spread on the plate and black spore grows out, selecting 1 × 1cm of Rhizopus nigricans hypha block with inoculating needle, transferring into a culture bottle containing potato glucose agar, culturing for 3 days until a large amount of black spore appears, washing Rhizopus nigricans with sterile water, collecting spore, and counting with a counting plate to obtain the product with spore density of 1 × 10 5 ～10 8 Per mL rhizopus nigricans spore suspension;
(2) selecting 200g of cleaned, peeled and cut fresh burdock root, adding the fresh burdock root into a 3L shake flask of 1000mL of potato glucose liquid culture medium, sterilizing by a wet method, and inoculating the fresh burdock root with the spore density of 1 multiplied by 10 according to the volume ratio of 2 percent 7 Putting the rhizopus nigricans spore suspension per mL into the conditions of 28 ℃ and 150rpm of oscillation speed for fermentation culture for 12 days to obtain burdock root fermentation liquor;
(3) filtering and clarifying burdock root fermentation liquid, concentrating the collected supernatant to 1/5 of the original volume, adding 95% ethanol, measuring by an alcohol meter until the final concentration of the ethanol is 75%, and standing overnight at 4 ℃; adding distilled water with the weight-volume ratio of 5-10 times of the collected alcohol precipitation substance, stirring for dissolving, centrifuging, removing precipitate, mixing the crude polysaccharide solution with a deproteinizing reagent (chloroform: n-butanol: 3: 1) according to the ratio of 4: adding the mixture into a conical flask according to the volume ratio of 1, stirring for 30min, pouring the mixture 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 with macroporous resin D301 or D301R chromatographic column until the crude polysaccharide solution is light yellow or colorless; concentrating the decolorized crude polysaccharide solution, filling into a dialysis bag with a molecular weight of 5000Da, placing the dialysis bag in distilled water, dialyzing for multiple times, and freeze-drying the solution to obtain a crude polysaccharide dry product;
dissolving a crude polysaccharide dry product in distilled water, passing through a 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 according to the sodium chloride concentration from small to large, collecting the eluent of the 0.02M tris-hydrochloric acid +0.3M sodium chloride solution to obtain crude burdock root polysaccharide RB3, performing molecular sieve exclusion agarose gel G100 and G200 chromatography, and collecting a sugar solution with a single peak to obtain purified burdock root polysaccharide RB 3.
The polysaccharide was extracted from the water-insoluble burdock root material not fermented by Rhizopus nigricans by hot water extraction, and then the polysaccharide RB3 of burdock root prepared by fermentation of Rhizopus nigricans of this example and the polysaccharide of the water-insoluble burdock root material not fermented by Rhizopus nigricans were passed through a DEAE fast flow chromatography column, and the results are shown in FIG. 1.
As can be seen from FIG. 1, the polysaccharide component extracted from the burdock root water-insoluble substance which is not fermented by Rhizopus nigricans is mainly neutral polysaccharide, and the polysaccharide component extracted from the burdock root water-insoluble substance fermented by Rhizopus nigricans in the embodiment is mainly acidic polysaccharide.
The burdock root polysaccharide RB3 prepared by using rhizopus nigricans for fermentation in the embodiment is prepared into a 5mg/ml solution, the solution is centrifuged at 12000rpm for 10min, the supernatant is filtered by a 0.22 mu m microporous filter membrane, then the sample is transferred into a 1.8ml sample injection bottle for High Performance Liquid Chromatography (HPLC),
the conditions are as follows: a 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 introduction amount: 20 mu l of the mixture; a detector: the difference detector RI-502, the result is shown in FIG. 2.
As can be seen from fig. 2, the weight average molecular mass of the burdock root polysaccharide RB3 prepared by rhizopus nigricans fermentation in this example is 140.0kDa, the sum of the molar content ratios is 63.3% with galactose and galacturonic acid as main components, 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 utilizing the rhizopus nigricans fermentation in the embodiment is shown in figure 3.
As can be seen from FIG. 3, RB3 is at 4000-400cm -1 There are a number of characteristic absorption peaks in the range. At 3417.88cm -1 The nearby broad absorption peak is the stretching vibration of the hydroxyl group OH. At 2928.37cm -1 The peak at (a) corresponds to the stretching vibration of the CH group. 1612.87cm -1 And 1415.02cm -1 The peak indicates the presence of carbonyl CO, suggesting the possible presence of uronic acid. 1200 and 1000cm -1 The absorption peaks in the range are attributable to the stretching vibration of C-O-C and C-O-H. Particularly at 1070.28cm -1 ,1042.66cm -1 And 896.41cm -1 The characteristic peaks 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 (a) indicates the presence of the sugar unit in the alpha configuration.
The analysis of the results of polysaccharide methylated glycitol acetyl ester (PMAA) of Burdock root polysaccharide RB3 prepared by Rhizopus nigricans fermentation in this example is shown in FIG. 4.
As can be seen from FIG. 4, the linkage type of each glycosidic bond in RB3, where the ratio of Gal1-4 is the sum of the reduction gain of galacturonic acid GalA1-4 and Gal1-4 itself.
In this example, the nuclear magnetic spectrum of the burdock root polysaccharide RB3 prepared by fermenting rhizopus nigricans is shown in figure 5.
As can be seen from FIG. 5, the signals in the hydrogen spectrogram A are mainly concentrated between 3.0 ppm and 5.5 ppm. Delta 3.2-4.0ppm is sugar ring proton signal, and the signal peaks of main terminal 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 distributed in a concentrated mode in a region of 4.3-5.5 ppm. Analysis of carbon spectrum B 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, the main anomeric carbon signal peaks delta 110.59 and 104.51 anomeric carbon regions can be seenMainly between delta 93 and delta 105. And 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 and 98.77 are distributed in the region of 60-85 ppm. 174.8ppm is carbonyl peak of uronic acid and 18.23ppm is C6 signal peak of rhamnose.
Through an HSQC map D, an anomeric carbon signal is delta 110.56, a corresponding anomeric hydrogen signal in the HSQC map is delta 5.17, and a signal combined with a COSY map F and H1-2 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 conclude that H1, H2, H3, H4, and H5a are δ 5.17, 4.13, 3.87, 3.99, and 3.76, respectively. The corresponding C1-C5 are 110.62, 82.62, 77.97, 85.22 and 62.64; therefore, the signal should be attributed to the glycosidic bond α -L-Araf- (1 →. another anomeric carbon signal is δ 104.48, the corresponding anomeric hydrogen signal in HSQC spectrum D is δ 4.4, the signal through COSY spectrum F, H1-2 is 4.4/3.28, H2-3 is 3.28/3.46, H3-4 is 3.46/3.21, H4-5a is 3.21/3.60, we can conclude that H1, H2, H3, H4, H5a are δ 4.4, 3.28, 3.46, 3.21, 3.60, the corresponding C1-C5 is 104.48, 74.66, 76.62, 83.4, 77.36, 62.3, respectively, and therefore, the signal should be attributed to the glycosidic bond β -D-Glcp- (1 →. approximate. Y and the binding of all glycosidic bonds according to similar laws,
RB3 backbone analysis: the anomeric hydrogen of → 2,4) - α -L-Rha- (1 → has a peak associated with H4 of → 4) - α -D-GalAp- (1 → in NOESY pattern E, indicating the presence of → 2,4) - α -L-Rha- (1 → 4) - α -D-GalAp- (1 →. The anomeric hydrogen of the glycosidic bond → 4) -alpha-D-GalAp- (1 → has a related signal peak with the H4 of the anomeric hydrogen; indicating the presence of the → 4) - α -D-GalAp- (1 → 4) - α -D-GalAp- (1 → linkage. Glycosidic bond → 4) - α -D-GalAp- (1 → anomeric hydrogen and → 4) - β -D-Galp- (1 → H4 have associated signal peaks; indicating the presence of the → 4) - α -D-GalAp- (1 → 4) - β -D-Galp- (1 → linkage. The anomeric hydrogen of → 4) -beta-D-Galp- (1 → has a relevant signal peak with the H4 of the anomeric hydrogen; indicating the presence of the → 4) - β -D-Galp- (1 → 4) - β -D-Galp- (1 → a linkage. → the anomeric hydrogen of → 4) - β -D-Galp- (1 → H3 of → 3,6) - β -D-Galp- (1 → has a relevant signal peak; indicating the presence of the → 4) - β -D-Galp- (1 → 3,6) - β -D-Galp- (1 → linkage. → 3,6) - β -D-Galp- (1 → anomeric hydrogen and → 2,4) - α -L-Rha- (1 → H4 have associated signal peaks; indicating the presence of the → 3,6) - β -D-Galp- (1 → 2,4) - α -L-Rha- (1 → linkage.
RB3 branch analysis: in the HMBC map C, a nuclear magnetic one-dimensional two-dimensional map is combined to attribute the glycosidic bond signal of the RB3 polysaccharide; the pattern of linkage of α -L-Araf- (1 → α -L-Araf- (1 → C5, which indicates the presence of α -L-Araf- (1 → 5) - α -L-Araf- (1 → the presence of the anomeric hydrogen of the glycosidic linkage → 5) - α -L-Araf- (1 → a signal peak associated with its own C5, the pattern of linkage of → 5) - α -L-Araf- (1 → 5) - α -L-Araf- (1 → the presence of the anomeric hydrogen of the glycosidic linkage → 5) - α -L-Araf- (1 → a signal peak associated with C5 of → 3,5- α -L-Araf- (1 → a signal peak indicating the presence of → 5) - α -L-Araf- (1 → 3,5) the linkage form of- α -L-Araf- (1 → g). The anomeric hydrogen of the glycosidic bond alpha-L-Araf- (1 → C3 of → 3,5) -alpha-L-Araf- (1 → has a signal peak related to the linkage of alpha-L-Araf- (1 → 3,5) -alpha-L-Araf- (1 → a peak related to the linkage of beta-D-Galp- (1 → 6) -beta-D-Galp- (1 → C6 of the → 1 → a peak related to the anomeric hydrogen of the beta-D-Galp- (1 → 6) -beta-D-Galp- (1 → C6 of the → a peak related to the anomeric hydrogen of the → 3,6) -beta-D-Galp- (1 → 3), 6) - β -D-Galp- (1 →. The anomeric carbon of beta-D-Glcp- (1 → and H4 of → 4) -beta-D-Glcp- (1 → have related peaks, which indicates that the anomeric carbon of beta-D-Glcp- (1 → 4) -beta-D-Glcp- (1 → 4 → is related to H4 of the anomeric carbon, and indicates that the anomeric carbon of beta-D-Glcp- (1 → 4) -beta-D-Glcp- (1 → 4 → 1 → 4) is related to H4.
In summary, the following steps: the burdock root polysaccharide RB3 mainly comprises monosaccharide, the molar ratio of rhamnose, arabinose, galactose and galacturonic acid is 1:1:2:2, and the content of other glycosidic bonds is too low to be ignored as a main reference. The main chain of the polysaccharide is mainly → 4) -alpha-L-Rha- (1 → 4) -alpha-D-GalAp- (1 → 4) -beta-D-Galp- (1 → composition, and the branched chains are connected to the main chain through → 2,4) -alpha-L-Rha- (1 → and → 3,6) -beta-D-Galp- (1 → O-2, O-6, respectively.
Example 2
A production process for preparing burdock root polysaccharide by utilizing rhizopus nigricans fermentation comprises the following steps:
(1) inoculating Rhizopus nigricans into a plate containing a potato glucose culture medium (PDA), culturing at 28 deg.C for 6 days until white hypha is spread on the plate and black spore grows out, selecting 1 × 1cm of Rhizopus nigricans hypha block with an inoculating needle, transferring into a culture bottle containing the potato glucose agar culture medium, culturing for 4 days until a large amount of black spore appears, washing Rhizopus nigricans thallus with sterile water in the culture bottle, collecting spore, counting with a counting plate to obtain the product with spore density of 1 × 10 5 Per mL rhizopus nigricans spore suspension;
(2) selecting fresh burdock root, treating by hot water extraction method to obtain water-insoluble burdock root, adding 250g of water-insoluble burdock root into a 3L triangular flask containing 1000mL of potato glucose liquid culture medium, performing wet sterilization, and inoculating with spore with density of 1 × 10 according to volume ratio of 5% V/V 5 Putting the rhizopus nigricans spore suspension per mL into the conditions of 28 ℃ and 150rpm of oscillation speed for fermentation culture for 15 days to obtain burdock root fermentation liquor; (3) filtering and clarifying burdock root fermentation liquor, concentrating the collected supernatant into 1/4 of the original volume, adding 95% ethanol, measuring by an alcohol meter until the final concentration of the ethanol is 75%, and standing overnight at 4 ℃; adding distilled water with the weight volume ratio of 5-10 times into the collected alcohol precipitation substance, stirring and dissolving, and removing precipitates after centrifugation to obtain a tawny crude polysaccharide solution; the crude polysaccharide aqueous solution was mixed with a deproteinizing reagent (chloroform: n-butanol ═ 3: 1) according to 3: adding the mixture into a conical flask according to the volume ratio of 1, stirring for 30min, pouring the mixture 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 with macroporous resin D301 or D301R chromatographic column until the crude polysaccharide solution is light yellow or colorless; concentrating the decolorized crude polysaccharide solution, filling into a dialysis bag with a molecular weight of 5000Da, placing the dialysis bag in distilled water, dialyzing for multiple times, and freeze-drying the solution to obtain a crude polysaccharide dry product;
dissolving the crude polysaccharide dry product in distilled water, passing through a 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 in sequence from small to large according to the sodium chloride concentration, collecting the eluent of 0.02M tris-hydrochloric acid +0.4M sodium chloride solution to obtain crude burdock root polysaccharide, and performing molecular sieve exclusion agarose gel G100 chromatography to obtain purified burdock root polysaccharide RB 3.
Wherein the process of obtaining the burdock root water-insoluble substance after the treatment by the hot water extraction method is as follows: selecting fresh burdock roots, inactivating enzyme in boiling water bath for 5-10min, peeling the burdock roots, cutting the burdock roots into slices, adding deionized water according to the feed liquid mass ratio of 1:10, leaching for 3 times under the condition of 80 ℃ and 60min, combining leaching liquor, filtering and leaching, centrifuging the filtrate, collecting precipitate, and obtaining the burdock root water-insoluble substance.
In the embodiment, the weight average molecular weight of the burdock root polysaccharide RB3 prepared by fermenting rhizopus nigricans is 149.5kDa, galactose and galacturonic acid are taken as main components, the sum of the molar content ratio is 50.5 percent, and the burdock root polysaccharide RB3 mainly comprises galactose, galacturonic acid, rhamnose, arabinose, glucuronic acid, glucose, mannose, xylose and fructose in the 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 utilizing rhizopus nigricans fermentation comprises the following steps:
(1) inoculating Rhizopus nigricans into a plate containing a potato glucose culture medium (PDA), culturing at 28 deg.C for 4 days until white hypha is spread on the plate and black spore grows out, selecting 1 × 1cm of Rhizopus nigricans hypha block with an inoculating needle, transferring into a culture bottle containing the potato glucose agar culture medium, culturing for 2 days until a large amount of black spore appears, washing Rhizopus nigricans thallus with sterile water in the culture bottle, collecting spore, counting with a counting plate to obtain the product with spore density of 1 × 10 8 Per mL rhizopus nigricans spore suspension;
(2) selecting fresh burdock root, treating by hot water extraction method to obtain water-insoluble burdock root, drying at 60 deg.C, pulverizing to obtain dry powder, adding 50g dry powder into 1000mL potato glucose liquid culture mediumThe spore density of the 3L triangular flask is 1 multiplied by 10 according to the volume ratio of 3 percent V/V after being sterilized by a wet method 8 Putting the rhizopus nigricans spore suspension per mL into the conditions of 28 ℃ and 150rpm of oscillation speed for fermentation culture for 7 days to obtain burdock root fermentation liquor;
(3) filtering and clarifying burdock root fermentation liquor, concentrating the collected supernatant into 1/4 of the original volume, adding 95% ethanol, measuring by an alcohol meter until the final concentration of the ethanol is 75%, and standing overnight at 4 ℃; adding distilled water with the weight volume ratio of 5-10 times into the collected alcohol precipitation substance, stirring and dissolving, and removing precipitates after centrifugation to obtain a tawny crude polysaccharide solution; the crude polysaccharide aqueous solution was mixed with a deproteinizing reagent (chloroform: n-butanol ═ 3: 1) according to 3: adding the mixture into a conical flask according to the volume ratio of 1, stirring for 30min, pouring the mixture 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 with macroporous resin D301 or D301R chromatographic column until the crude polysaccharide solution is light yellow or colorless; concentrating the decolorized crude polysaccharide solution, filling into a dialysis bag with a molecular weight of 5000Da, placing the dialysis bag in distilled water, dialyzing for multiple times, and freeze-drying the solution to obtain a crude polysaccharide dry product;
dissolving the crude polysaccharide dry product in distilled water, passing through a 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 in sequence from small to large according to the concentration of the sodium chloride solution, collecting the eluent of the 0.02M tris-hydrochloric acid +0.4M sodium chloride solution to obtain crude burdock root polysaccharide, and performing molecular sieve exclusion agarose gel G100 chromatography to obtain purified burdock root polysaccharide RB 3.
In the embodiment, the weight average molecular weight of the burdock root polysaccharide RB3 prepared by fermenting rhizopus nigricans is 128.1kDa, galactose and galacturonic acid are taken as main components, the sum of the molar content ratio is 54.7 percent, and the burdock root polysaccharide RB3 mainly comprises galactose, galacturonic acid, rhamnose, arabinose, glucuronic acid, mannose and xylose in the molar ratio of 29.8:24.9:19.3:13.5:4.2:7.1: 1.2.
Example 4 cell assay
1. Burdock root polysaccharide RB3 prepared in example 1 was used to perform cytotoxicity test on human normal liver cells LO2
The specific method comprises the following steps:
culturing human normal liver cell LO2 with RPMI1640 basic culture medium, 10% fetal calf serum and 1% double antibody, placing in a constant temperature incubator containing 5% carbon dioxide, and culturing at 37 deg.C. After digesting the cells, the ratio of 5X 10 3 And (2) paving each well in a 96-well plate, dissolving RB3 in RPMI160 culture solution, filtering and sterilizing, treating cells with RB3 with different concentration gradients of 0-1500 mu g/mL for 24 hours, removing the culture solution, adding CCK-8 reagent, reacting at 37 ℃ for 1 hour, and detecting the absorbance at 540nm by using an enzyme reader.
As can be seen from FIG. 6, the Burdock root polysaccharide RB3 with the concentration of 100-.
2. The specific method for carrying out the immunocompetence experiment on the mouse macrophage RAW264.7 by the burdock root polysaccharide RB3 prepared in the example 1 is as follows:
culturing mouse macrophage RAW264.7 with RPMI1640 basic culture solution, 10% fetal calf serum and 1% double antibody, placing in a constant temperature incubator containing 5% carbon dioxide, and culturing at 37 deg.C. After digesting the cells, the ratio of 5X 10 3 And paving the cells in a 96-well plate, dissolving RB3 in RPMI160 culture solution, filtering and sterilizing, treating the cells with RB3 with different concentration gradients of 0-900 mu g/mL, sucking out the culture solution after 24 hours, adding CCK-8 reagent, reacting for 1 hour at 37 ℃, and detecting the influence of RB3 on macrophage proliferation by using an enzyme labeling instrument to detect the absorbance at 540 nm. In addition, the culture medium was collected and centrifuged at 5000 Xg, and the contents of cytokines such as NO, IL- β, IL-6, TNF- α and the like were measured.
As can be seen from FIG. 7, compared with the control group, the Burdock root polysaccharide RB3 with the concentration of 50-900 μ g/mL can remarkably promote macrophage proliferation and is in direct proportion to the polysaccharide concentration, and the proliferation rate can reach 135% at most.
As shown in FIG. 8, compared with the control group, the Burdock root polysaccharide RB3 with the concentration of 50-900 μ g/mL can also enhance the phagocytic activity of macrophages and induce the secretion of cytokines such as NO, IL- β, IL-6, TNF- α and the like of the macrophages.
3. Experiment of proliferation of tumor cells by Burdock root polysaccharide RB3 prepared in example 1
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 for 24h by RPMI1640 basic culture solution, 10 percent fetal bovine serum and 1 percent double antibody. Dissolving Arctium lappa root polysaccharide RB3 in RPMI160 culture solution, filtering for sterilization, treating cells with prepared culture solution containing RB3 with different concentrations for 48h, and detecting the survival rate of cancer cells with CCK-8 reagent.
Experimental results show that the burdock root polysaccharide RB3 has a good inhibition effect on the proliferation of the tumor cells and presents an obvious dose dependence relationship, and the IC50 doses of the burdock root polysaccharide RB3 on the tumor cells are 753, 521, 243 and 855 mug/mL in sequence.
Example 5 results of in vivo tumor-bearing mice
Injecting a proper amount of S180 cells under the right axilla of the Balb/c female mouse to establish an S180 tumor-bearing mouse model. Using the Burdock root polysaccharide RB3 prepared in example 1, a Burdock root polysaccharide RB3 low dose group (50mg/kg/d), a medium dose group (100mg/kg/d), a high dose group (150mg/kg/d), a 5-fluorouracil group (20mg/kg/d), and a combined 5-fluorouracil (20mg/kg/d) + medium dose group (100mg/kg/d) were set. The administration mode is that the number of the mice is 0.2ml/d, and the negative control groups 1-2 are the burdock root polysaccharide (500mg/kg/d) extracted by distilled water and hot water with the same dose for the stomach filling of the modeling mice.
Selecting mice of about 8 weeks, adaptively feeding common feed for 1 week, injecting a proper amount of S180 cells into right axilla, inoculating for about one week, modeling successfully, and continuously taking medicine for 14 days. After overnight fasting without water deprivation for the last day and weighing the body weight the following day, mice were sacrificed by cervical dislocation, tumors were dissected 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 (50mg/kg/d), the medium dose group (100mg/kg/d), the high dose group (150mg/kg/d) and the 5-fluorouracil group (20mg/kg/d) of Burdock root polysaccharide RB3 were 34%, 60%, 81% and 86%, respectively, as compared with the average tumor volume of the negative control group. Wherein, the difference between the medium dose group and the high dose group has statistical significance (p is less than 0.05). In addition, the tumor inhibition effect of the 5-fluorouracil and medium-dose combination group is better than that of the single 5-fluorouracil group, and the combined 5-fluorouracil and medium-dose combination group has better recovery effect on mouse body weight, immune tissues and the like. But the burdock root polysaccharide extracted by hot water has no obvious in-vivo anti-tumor activity.
Claims (10)
1. The burdock root polysaccharide prepared by using rhizopus nigricans for fermentation is characterized in that the weight average molecular weight of the burdock root polysaccharide is 5 multiplied by 10 4 ～1×10 6 Da, the molar content of galactose and galacturonic acid in the burdock root polysaccharide is more than 50%.
2. The production process for preparing burdock root polysaccharide by using rhizopus nigricans fermentation in claim 1, which is characterized by comprising the following steps:
(1) inoculating Rhizopus nigricans strains into a potato glucose agar culture medium, culturing at 26-30 ℃ for 4-6 days until white hyphae are fully paved and black spores are grown, selecting Rhizopus nigricans hypha blocks, transferring into the potato glucose agar culture medium, continuously culturing for 2-4 days until a large number of black spores appear, washing the Rhizopus nigricans thalli with sterile water, and collecting the spores to obtain a Rhizopus nigricans spore suspension;
(2) selecting fresh burdock roots or burdock root water-insoluble substances or burdock root water-insoluble dry powder, adding the fresh burdock roots or burdock root water-insoluble dry powder into a shake flask of a potato glucose liquid culture medium, performing wet sterilization, then inoculating rhizopus nigricans spore suspension according to the volume ratio of 0.5-5%, and then performing fermentation culture for 7-15 days under the conditions that the temperature is 25-30 ℃ and the oscillation speed is 120-200rpm, so as to obtain burdock root fermentation liquid;
(3) filtering and clarifying burdock root fermentation liquor, concentrating supernatant, precipitating with ethanol, redissolving, deproteinizing, decolorizing, dialyzing, and 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.
3. The process according to claim 2, wherein in the step (1), the size of the Rhizopus nigricans mycelial block is 1 x 1 cm; the density of the rhizopus nigricans spores in the rhizopus nigricans spore suspension is 1 multiplied by 10 5 ～10 8 one/mL.
4. The process according to claim 2, wherein in step (2), the Rhizopus nigricans spore suspension is inoculated in an amount of 2% by volume of potato dextrose broth.
5. The production process of claim 2, wherein in the step (2), the fresh burdock roots are cleaned, peeled and cut, and the weight-to-volume ratio of the fresh burdock roots to the potato dextrose liquid culture medium is (1-4): 10, unit is: g/mL.
6. The production process according to claim 2, wherein in the step (2), the burdock root water-insoluble substance is obtained by treating fresh burdock roots through a hot water extraction method, and the weight volume ratio of the burdock root water-insoluble substance to a potato dextrose liquid culture medium is (1-4): 10, unit is: g/mL.
7. The production process according to claim 2, wherein in the step (2), the burdock root water-insoluble dry powder is obtained by drying and crushing burdock root water-insoluble at 50-70 ℃, and the weight-volume ratio of the burdock root water-insoluble dry powder to the potato dextrose broth is (1-5): 100, unit is: g/mL.
8. The process according to claim 2, wherein in the step (3), the anion-exchange DEAE fast flow chromatography is carried out by the following method: the burdock root polysaccharide RB3 prepared by rhizopus nigricans fermentation is obtained by adopting 0.02M tris-hydrochloric acid, 0.02M tris-hydrochloric acid and 0.1M sodium chloride solution, 0.02M tris-hydrochloric acid and 0.2M sodium chloride solution, 0.02M tris-hydrochloric acid and 0.3M sodium chloride solution, 0.02M tris-hydrochloric acid and 0.4M sodium chloride solution and 0.02M tris-hydrochloric acid and 0.5M sodium chloride solution to elute according to the sodium chloride concentration from small to large in sequence and collecting the eluent of 0.02M tris-hydrochloric acid and 0.3M sodium chloride solution.
9. The use of the burdock root polysaccharide of claim 1 in the preparation of immunomodulatory antitumor drugs or foods.
10. The use of claim 9, wherein the burdock root polysaccharide is used in combination with 5-fluorouracil, the burdock root polysaccharide is used in an amount of 50-150 mg/kg/d, and the 5-fluorouracil is used in an amount of 15-25 mg/kg/d.
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