CN116617247B - Roughhaired holly root polysaccharide prepared by organic ultrafiltration membrane purification and separation and application thereof - Google Patents
Roughhaired holly root polysaccharide prepared by organic ultrafiltration membrane purification and separation and application thereof Download PDFInfo
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- CN116617247B CN116617247B CN202310913208.1A CN202310913208A CN116617247B CN 116617247 B CN116617247 B CN 116617247B CN 202310913208 A CN202310913208 A CN 202310913208A CN 116617247 B CN116617247 B CN 116617247B
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- holly root
- roughhaired holly
- root polysaccharide
- polysaccharide
- roughhaired
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Classifications
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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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- 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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/899—Poaceae or Gramineae (Grass family), e.g. bamboo, corn or sugar cane
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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
- A61P37/04—Immunostimulants
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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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides roughhaired holly root polysaccharide prepared by organic ultrafiltration membrane purification and separation and application thereof, and belongs to the technical field of polysaccharide. The roughhaired holly root polysaccharide with different molecular weights is obtained through the purification and separation of the organic ultrafiltration membrane and the macroporous resin, and experiments show that the roughhaired holly root polysaccharide prepared by the invention can effectively inhibit the proliferation of various tumor cells and can effectively inhibit iron death caused by inflammation. Secondly, through cell mouse experiments, the roughhaired holly root polysaccharide prepared by the invention can effectively enhance the immunity of mice. Therefore, the invention widens the application range of the roughhaired holly root polysaccharide and provides a new efficient and safe drug selection for the treatment of diseases.
Description
Technical Field
The invention belongs to the technical field of polysaccharide, and particularly relates to roughhaired holly root polysaccharide prepared by organic ultrafiltration membrane purification and separation and application thereof.
Background
The root of Ilicis Asprellae is root of Ilicis Asprellae (also called as semen Setariae, rice washing spike) belonging to Gramineae. The roughhaired holly is a common traditional Chinese medicine, is widely used in the field of traditional Chinese medicines, and has higher medicinal value. The radix Ilicis Asprellae has effects of clearing heat and promoting diuresis, invigorating spleen and stomach, inducing diuresis and relieving swelling. The traditional Chinese medicine is considered to be sweet in taste and neutral in nature, and can clear heat and promote diuresis, invigorate spleen and stomach, promote urination and reduce edema. Is mainly used for treating damp-heat syndrome, damp-warm syndrome, spleen and stomach dampness stagnation syndrome and the like.
The 104 chemical components of the holly have been reported to be separated and identified, and the number and content of the chemical components are mainly triterpenoid saponins, especially the ursane (ursane), and the chemical components also contain chlorogenic acid, flavone, phenylpropanoid, lignans, a small amount of steroids and other types. However, there is little research on roughhaired holly root polysaccharide, and its function is still yet to be further developed.
Iron death is a novel programmed cell death mode induced by iron-dependent lipid peroxidation and plays a very important role in pathophysiological processes such as inflammation, cancer, neurodegenerative diseases, organ injury and the like. Macrophage inflammatory response is closely related to iron death. At present, no related research report exists on the regulation and control effect of roughhaired holly root polysaccharide on macrophage iron death.
Secondly, there are few reports on the role of roughhaired holly root polysaccharide in tumor inhibition, so that the role of roughhaired holly root polysaccharide in tumor inhibition is studied.
Disclosure of Invention
The invention aims to provide roughhaired holly root polysaccharide prepared by organic ultrafiltration membrane purification and separation and application thereof, thereby widening the application range of roughhaired holly root polysaccharide and providing a safe and reliable medicament for treating tumor diseases and improving immunity.
In order to achieve the above purpose, the present invention provides the following technical solutions:
firstly, the invention provides an application of roughhaired holly root polysaccharide prepared by organic ultrafiltration membrane purification and separation in preparation of tumor cell proliferation inhibition drugs.
Preferably, the roughhaired holly root polysaccharide is divided into 3 types, namely roughhaired holly root polysaccharide 1, roughhaired holly root polysaccharide 2 and roughhaired holly root polysaccharide 3;
the monosaccharide composition of the roughhaired holly root polysaccharide 1 is 1.04% of Rha,0.70% of Ara,3.05% of Gal,78.98% of Glc,1.29% of Man,4.65% of Gal-UA,5.80% of Gul-UA,1.28% of Glc-UA and 3.22% of Man-UA; the monosaccharide composition of the roughhaired holly root polysaccharide 2 is 0.73% Rha,0.45% Gal,78.53% Glc,5.75% Gal-UA,8.43% Gul-UA,1.73% Glc-UA and 4.38% Man-UA; the monosaccharide composition of the roughhaired holly root polysaccharide 3 is 1.70% Rha,2.14% Ara,3.92% Gal,87.45% Glc,0.72% Man,1.29% Gal-UA,1.93% Gul-UA and 0.84% Glc-UA.
Preferably, the preparation method of the roughhaired holly root polysaccharide comprises the following steps:
(1) Processing radix Ilicis Asprellae into residue, and reflux extracting with ethanol to remove oil;
(2) Adding 20 times of distilled water in water bath at 80deg.C, collecting supernatant, repeating operation for 2 times again, and mixing the 3 supernatants to obtain radix Ilicis Asprellae polysaccharide extractive solution;
(3) Concentrating the roughhaired holly root polysaccharide extract by rotary evaporation at 70 ℃ to 20% of the original volume, adding 4 times of ethanol to make the ethanol concentration reach 80%, standing overnight at 4 ℃, centrifuging and taking out precipitate to obtain roughhaired holly root polysaccharide;
(4) Preparing the roughhaired holly root crude polysaccharide into a roughhaired holly root crude polysaccharide solution with distilled water at a concentration of 0.1g/mL, and mixing with chloroform: n-butanol=4:1 volume ratio to configure Sevag reagent;
(5) Mixing the roughhaired holly root crude polysaccharide solution and Sevag reagent according to the volume ratio of 5:1, and repeating the operation for 8 times to remove protein;
(6) The roughhaired holly root crude polysaccharide solution with protein removed and AB-8 macroporous resin are mixed according to the following ratio of 1:4, adding the mixture into a constant temperature oscillator for oscillating for 16 hours to remove the pigment of the polysaccharide;
(7) Filtering with 10kDa filter membrane, removing filtrate, passing the retentate through 50kDa filter membrane to obtain filtrate which is radix Ilicis Asprellae polysaccharide solution 1 with molecular weight of 10-50kDa, passing the retentate through 100kDa filter membrane to obtain filtrate which is radix Ilicis Asprellae polysaccharide solution 2 with molecular weight of 50-100kDa, and obtaining retentate which is radix Ilicis Asprellae polysaccharide solution 3 with molecular weight of more than 100 kDa.
(8) And freeze-drying the roughhaired holly root polysaccharide solution 1, the roughhaired holly root polysaccharide solution 2 and the roughhaired holly root polysaccharide solution 3 to obtain roughhaired holly root polysaccharide 1, roughhaired holly root polysaccharide 2 and roughhaired holly root polysaccharide 3.
Preferably, the tumor cells include lung cancer cells, liver cancer cells, breast cancer cells and colorectal cancer cells.
Secondly, the invention provides application of roughhaired holly root polysaccharide prepared by purifying and separating by an organic ultrafiltration membrane in preparation of macrophage iron death caused by inflammation inhibition.
Preferably, the roughhaired holly root polysaccharide is divided into 3 types, namely roughhaired holly root polysaccharide 1, roughhaired holly root polysaccharide 2 and roughhaired holly root polysaccharide 3;
the monosaccharide composition of the roughhaired holly root polysaccharide 1 is 1.04% of Rha,0.70% of Ara,3.05% of Gal,78.98% of Glc,1.29% of Man,4.65% of Gal-UA,5.80% of Gul-UA,1.28% of Glc-UA and 3.22% of Man-UA;
the monosaccharide composition of the roughhaired holly root polysaccharide 2 is 0.73% Rha,0.45% Gal,78.53% Glc,5.75% Gal-UA,8.43% Gul-UA,1.73% Glc-UA and 4.38% Man-UA;
the monosaccharide composition of the roughhaired holly root polysaccharide 3 is 1.70% Rha,2.14% Ara,3.92% Gal,87.45% Glc,0.72% Man,1.29% Gal-UA,1.93% Gul-UA and 0.84% Glc-UA.
Preferably, the roughhaired holly root polysaccharide is prepared by a preparation method of the roughhaired holly root polysaccharide.
Secondly, the invention provides application of roughhaired holly root polysaccharide prepared by organic ultrafiltration membrane purification and separation in preparation of immunity enhancing drugs, wherein the roughhaired holly root polysaccharide increases the expression level of TNF-alpha and IL-6 in blood and improves the spleen index of treated individuals.
Preferably, the roughhaired holly root polysaccharide is divided into 3 types, namely roughhaired holly root polysaccharide 1, roughhaired holly root polysaccharide 2 and roughhaired holly root polysaccharide 3;
the monosaccharide composition of the roughhaired holly root polysaccharide 1 is 1.04% of Rha,0.70% of Ara,3.05% of Gal,78.98% of Glc,1.29% of Man,4.65% of Gal-UA,5.80% of Gul-UA,1.28% of Glc-UA and 3.22% of Man-UA;
the monosaccharide composition of the roughhaired holly root polysaccharide 2 is 0.73% Rha,0.45% Gal,78.53% Glc,5.75% Gal-UA,8.43% Gul-UA,1.73% Glc-UA and 4.38% Man-UA;
the monosaccharide composition of the roughhaired holly root polysaccharide 3 is 1.70% Rha,2.14% Ara,3.92% Gal,87.45% Glc,0.72% Man,1.29% Gal-UA,1.93% Gul-UA and 0.84% Glc-UA.
Preferably, the roughhaired holly root polysaccharide is prepared by a preparation method of the roughhaired holly root polysaccharide.
Secondly, the invention provides application of roughhaired holly root polysaccharide prepared by organic ultrafiltration membrane purification and separation in preparation of an accelerant for promoting expression of TNF-alpha and IL-6 in blood of immunocompromised individuals, wherein roughhaired holly root polysaccharide is divided into 3 types, namely roughhaired holly root polysaccharide 1, roughhaired holly root polysaccharide 2 and roughhaired holly root polysaccharide 3;
the monosaccharide composition of the roughhaired holly root polysaccharide 1 is 1.04% of Rha,0.70% of Ara,3.05% of Gal,78.98% of Glc,1.29% of Man,4.65% of Gal-UA,5.80% of Gul-UA,1.28% of Glc-UA and 3.22% of Man-UA;
the monosaccharide composition of the roughhaired holly root polysaccharide 2 is 0.73% Rha,0.45% Gal,78.53% Glc,5.75% Gal-UA,8.43% Gul-UA,1.73% Glc-UA and 4.38% Man-UA;
the monosaccharide composition of the roughhaired holly root polysaccharide 3 is 1.70% Rha,2.14% Ara,3.92% Gal,87.45% Glc,0.72% Man,1.29% Gal-UA,1.93% Gul-UA and 0.84% Glc-UA.
The invention has the beneficial effects that:
the invention prepares 3 roughhaired holly root polysaccharide through organic ultrafiltration membrane purification and separation, and meanwhile, through cell experiments, the roughhaired holly root polysaccharide prepared by the invention can effectively inhibit various tumor cells, and can reduce macrophage iron death caused by inflammation. Secondly, through cell mouse experiments, the roughhaired holly root polysaccharide prepared by the invention can effectively enhance the immunity of mice. Therefore, the invention widens the application range of the roughhaired holly root polysaccharide and provides a new efficient and safe drug selection for the treatment of diseases.
Drawings
FIG. 1 is an infrared absorption spectrum of holly root polysaccharides of different molecular weights;
FIG. 2 is a graph showing the results of a monosaccharide composition analysis of different molecular weight Ilicis Asprellae root polysaccharides;
FIG. 3 is a scanning electron microscope image of holly root polysaccharides of different molecular weights;
FIG. 3 (a) is an IAPS1 electron microscope scan at 5000 Xmagnification; fig. 3 (b) is an IAPS2 electron microscope scan at 5000 x magnification; fig. 3 (c) is an IAPS3 electron microscope scan at 5000 x magnification; fig. 3 (d) shows an IAPS1 electron microscope scan at 10000 times magnification; fig. 3 (e) shows an IAPS2 electron microscope scan at 10000 times magnification; fig. 3 (f) shows an IAPS3 electron microscope scan at 10000 times magnification;
FIG. 4 is a graph showing the effect of different molecular weight roughhaired holly root polysaccharides on LPS-induced fluorescent expression of macrophage ROS;
FIG. 5 is a graph showing the effect of different molecular weight roughhaired holly root polysaccharides on the weight loss rate of mice;
FIG. 6 is a graph of spleen index of different molecular weight roughhaired holly root polysaccharides in animal experiments;
FIG. 7 is a graph showing TNF- α content in blood after treatment with holly root polysaccharides of different molecular weights;
FIG. 8 is a graph showing the IL-6 content of blood after treatment with holly root polysaccharides of different molecular weights;
FIG. 9 is a view of a spleen HE stained section after treatment with Ilicis Asprellae root polysaccharide IAPS 1;
FIG. 9 (a) is a view of a spleen HE stained section of the Control group; fig. 9 (b) is a map of spleen HE staining sections of CTX group; FIG. 9 (c) is a view of spleen HE staining section of L-IAPS1 group; FIG. 9 (d) is a view of the spleen HE stained section of the H-IAPS1 group; FIG. 9 (e) is a view of a Control group of lymphoHE-stained sections; FIG. 9 (f) is a view of a lymphoHE-stained section of the CTX group; FIG. 9 (g) is a view of a lymphoHE-stained section of the L-IAPS1 group; FIG. 9 (H) is a view of a lymphoHE-stained section of the H-IAPS1 group;
FIG. 10 is a view of a spleen HE stained section after treatment with Ilicis Asprellae root polysaccharide IAPS 2;
FIG. 10 (a) is a view of a spleen HE stained section of the Control group; fig. 10 (b) is a map of spleen HE staining sections of CTX group; FIG. 10 (c) is a view of spleen HE staining sections of the L-IAPS2 group; FIG. 10 (d) is a view of the spleen HE stained section of the H-IAPS2 group; FIG. 10 (e) is a view of a Control group of lymphoHE-stained sections; FIG. 10 (f) is a view of a lymphoHE-stained section of the CTX group; FIG. 10 (g) is a view of a lymphoHE-stained section of the L-IAPS2 group; FIG. 10 (H) is a view of a lymphoHE-stained section of the H-IAPS2 group;
FIG. 11 is a view of a spleen HE stained section after treatment with Ilicis Asprellae root polysaccharide IAPS 3;
FIG. 11 (a) is a view of a spleen HE stained section of the Control group; fig. 11 (b) is a map of spleen HE staining sections of CTX group; FIG. 11 (c) is a view of spleen HE staining sections of the L-IAPS3 group; FIG. 11 (d) is a view of the spleen HE stained section of the H-IAPS3 group; FIG. 11 (e) is a view of a Control group of lymphoHE-stained sections; FIG. 11 (f) is a view of a lymphoHE-stained section of the CTX group; FIG. 11 (g) is a view of a lymphoHE-stained section of the L-IAPS3 group; FIG. 11 (H) is a view of a lymphoHE-stained section of the H-IAPS3 group.
Detailed Description
The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent.
Example 1: preparation of roughhaired Holly root polysaccharide
(1) Treating 500g of roughhaired holly root into slag by a pulverizer, and extracting 3 times by reflux with 95% ethanol for 3L to remove impurities such as grease;
(2) Adding 10L distilled water in 80deg.C water bath, collecting supernatant, repeating operation for 2 times again, and mixing the supernatants for 3 times to obtain radix Ilicis Asprellae polysaccharide extractive solution;
(3) Concentrating radix Ilicis Asprellae polysaccharide extractive solution at 70deg.C by rotary evaporation to 20% of original volume, adding 4 times volume of ethanol to 80%, standing overnight at 4deg.C, centrifuging to obtain precipitate to obtain radix Ilicis Asprellae crude polysaccharide;
(4) The roughhaired holly root polysaccharide is prepared into 500mL of roughhaired holly root polysaccharide solution with 0.1g/mL by distilled water according to chloroform: n-butanol=4:1 volume ratio to configure Sevag reagent;
(5) Mixing roughhaired holly root crude polysaccharide solution and Sevag reagent according to the volume ratio of 5:1, and repeating the operation for 8 times to remove protein;
(6) The roughhaired holly root crude polysaccharide solution with protein removed and AB-8 macroporous resin are mixed according to the following ratio of 1:4, adding the mixture into a constant temperature oscillator for oscillating for 16 hours to remove the pigment of the polysaccharide;
(7) Filtering with 10kDa filter membrane, removing filtrate, passing the retentate through 50kDa filter membrane to obtain filtrate which is radix Ilicis Asprellae polysaccharide solution 1 with molecular weight of 10-50kDa, passing the retentate through 100kDa filter membrane to obtain filtrate which is radix Ilicis Asprellae polysaccharide solution 2 with molecular weight of 50-100kDa, and obtaining retentate which is radix Ilicis Asprellae polysaccharide solution 3 with molecular weight of more than 100 kDa.
(8) And freeze-drying the roughhaired holly root polysaccharide solution 1, the roughhaired holly root polysaccharide solution 2 and the roughhaired holly root polysaccharide solution 3 to obtain roughhaired holly root polysaccharide 1, roughhaired holly root polysaccharide 2 and roughhaired holly root polysaccharide 3.
Example 2: detection of polysaccharide structures by infrared spectroscopy
The spectral structure is detected by infrared spectrum, the structure is shown in figure 1, and as can be seen from figure 1, 3309.16 cm -1 A wide and strong absorption band is a stretching vibration peak of hydroxyl, 1613.27 cm -1 The absorption band at this point may be derived from absorbed water at 1405.29 cm- 1 The nearby band is caused by the distorted vibration of C-H. For IAPS2 and IAPS3, 1125.41, 1091.91 and 1026.74 cm -1 The absorption peak at this point indicates the presence of a pyranose. Furthermore, 864.22, 757.57 and 657.11 cm -1 The absorption peak at this point is a characteristic absorption peak of the glucopyranose derivative.
Example 3: ion chromatography for detecting polysaccharide composition
Fucose, murine Li Tang, arabinose, galactose, glucose, xylose, mannose, fructose, galacturonic acid glucose, glucuronic acid and mannuronic acid were used as monosaccharide standards
(1) Sample 5 mg was weighed and 1mL of 2M trifluoroacetic acid (TFA, 3 mol/L) was added and heated at 121℃for 3 hours;
(2) Then, it was dried under a nitrogen stream. Subsequently, 50mL of water was added for vortex mixing;
(3) After filtration, the supernatants were analyzed using HPIC equipped with Dionex ™ CarboPac ™ PA20 (150 x 3.0mm,10 μm) liquid chromatography column and electrochemical detector;
(4) 0.1M NaOH, 0.1M NaOH and 0.2M NaAc were used as mobile phases, the flow rate was set to 0.5 mL/min, the sample injection amount was 5uL, and the column temperature was 30 ℃;
(5) The monosaccharide standard solution is treated by the same method, and the molar ratio is calculated according to the molar mass of the monosaccharide;
(6) Monosaccharide composition of the holly root polysaccharide samples of different molecular weights were analyzed by HPIC, the composition was determined using IC and by matching the retention time in the chromatographic curve with the retention time of the monosaccharide standard, and the results are shown in table 1 and fig. 2.
TABLE 1 analysis of the composition of the polysaccharide monosaccharides from Ilicis Asprellae root of different molecular weights
| Group | Rha | Ara | Gal | Glc | Man | Gal-UA | Gul-UA | Glc-UA | Man-UA |
| IAPS1 | 1.04% | 0.70% | 3.05% | 78.98% | 1.29% | 4.65% | 5.80% | 1.28% | 3.22% |
| IAPS2 | 0.73% | 0.00% | 0.45% | 78.53% | 0.00% | 5.75% | 8.43% | 1.73% | 4.38% |
| IAPS3 | 1.70% | 2.14% | 3.92% | 87.45% | 0.72% | 1.29% | 1.93% | 0.84% | 0.00% |
As can be seen from Table 1 and FIG. 2, IAPS1 has the greatest amounts of Glc and Gul-UA, which account for 78.98% and 5.8%; the largest amounts of Glc and Gul-UA in IAPS2 were 78.53% and 8.43% and the largest amounts of Glc and Gal in IAPS3 were 87.45% and 3.92%.
Example 4: SEM examination of polysaccharide results morphology
(1) The surface morphology features of 3 different molecular weight roughhaired holly root polysaccharides were observed using the Hitachi S-3400N-II SEM system at an accelerating voltage of 20 kV;
(2) The sample was fixed on the sample holder by means of a double-sided carbon tape, and then sputtered with platinum powder using an ion sputter coater, and the result was shown in fig. 3.
As can be seen from fig. 3, IAPS1 is in the form of elongated rice grains ((a) in fig. 3, and (b) in fig. 3), IAPS2 polysaccharide is in the form of clustered spheres ((c) in fig. 3, and (d) in fig. 3), and IAPS3 is in the form of relatively full rice grains ((e) in fig. 3, and (f) in fig. 3).
Example 5: inhibition of roughhaired holly root polysaccharide on proliferation of different tumor cells and normal cells
(1) The roughhaired holly root polysaccharide 1-3 (IAPS 1-3) was formulated as a 100 μg/ml roughhaired holly root polysaccharide solution.
(2) Selecting logarithmic growth phase cell, and adjusting cell density of 4 cancer cells (lung cancer cell A549, liver cancer cell HepG2, human breast cancer cell MCF-7, colon cancer cell SW 480) to 1.5X10 4 2 normal cells (hepatocyte L-02, embryonic lung fibroblast HFL-1) density was 2.0X10/mL 4 Per mL, adding cell suspension into 96-hole culture plates at 100 [ mu ] L/hole, arranging 3 parallel cells in each group, and placing in37. Culturing in a constant temperature and humidity incubator with 5% CO2 at the temperature;
(3) After 24 mu L of samples with different concentrations are added after h, after the samples are continuously cultured for 24 hours, 10 mu L of CCK-8 solution is added into each hole, the incubator is incubated for 4h, and the absorbance at 450nm is measured by using a microplate reader. The median inhibitory concentration IC50 values were finally calculated and the experimental results are shown in table 2.
TABLE 2 inhibition of proliferation of different tumor cells and Normal cells by roughhaired holly root polysaccharide
| Cells | IAPS1IC50 (µg/mL) | IAPS2IC50 (µg/mL) | IAPS3IC50 (µg/mL) |
| A549 | 105.6 | 147.8 | 180.3 |
| HepG2 | 231.3 | 262.2 | 295.9 |
| SW480 | 525.4 | 552.4 | 580.8 |
| MCF-7 | 893.2 | 921.4 | 956.2 |
| L-02 | 922.3 | 955.7 | 989.4 |
| HFL-1 | 929.9 | 960.3 | 991.5 |
From Table 2, the IC50 values of human tumor cells and normal cells are, in order from large to small, embryonal lung fibroblast HFL-1, hepatic cell L02, breast cancer cell MCF-7, colon cancer cell SW480, liver cancer cell HepG2, lung cancer cell A549; as can be seen, the inhibition effect of roughhaired holly root polysaccharide on tumor cell proliferation is greater than that of normal cells, the activity of roughhaired holly root polysaccharide on lung cancer cells A375 is maximum, the IC50 value is 105.6 mug/mL, the activity of roughhaired holly root polysaccharide on embryo lung fibroblast HFL-1 is minimum, the IC50 value is 929.9 mug/mL, and the cell selectivity is shown. And the IC50 values of the same cell show IAPS1 < IAPS2 < IAPS3, which shows that the low molecular weight IAPS1 has better tumor inhibition activity.
Example 6: detection of the Effect of Baeckea root polysaccharide on iron death
(1) Logarithmic growth of macrophages at 1.5X10 5 Density of wells/well was inoculated in 24 well plates and after 24h cells had attached, the experimental group had been pretreated with 1-3 roughhaired holly root polysaccharide 1 h and then had 1 mg/mL of LPS added to stimulate cells 24 h;
(2) After the drug treatment is finished, 500 mu L of serum-free culture solution containing 2.5 mu mol/L C BODIPY581/59 is added, the culture solution is incubated for 30 min at 37 ℃ in a dark place, and the cell lipid active oxygen fluorescence expression condition is observed under a laser confocal fluorescence microscope, and the result is shown in FIG. 4.
In fig. 4, the white color surrounding the cells is the ROS after oxidation, there are more redox cells following dry roughhaired holly root polysaccharide than the blank group, indicating a significant decrease in the degree of cellular oxidation, and the high dose group has a higher degree of redox than the low dose, IAPS1 has a higher degree of redox than IAPS2 and IAPS3. The roughhaired holly root polysaccharide has an inhibiting effect on inflammatory macrophage iron death induced by LPS, and IAPS1 has better activity of inhibiting iron death.
Example 7: detection of Effect of roughhaired Holly root polysaccharide on in vitro immunocompetence
(1) Mice (SPF-class Balb/C mice purchased from Jinan Pengyue laboratory animal breeding Co., ltd. (Shandong, china)) were opened in cages, divided into 8 groups of 5 mice each, and cultured under standard conditions (temperature 24.+ -. 2 ℃ C., relative humidity 50% -60% for 12 hours, and a lighting period was provided with conventional pellet feed and tap water). All experiments related to animals are strictly carried out in the field of nursing and use of experimental animals;
(2) The composition is divided into a control group, a cyclophosphamide model group (80 mg/kg), a 10-50kDa roughhaired holly root polysaccharide low dose group (100 mg/kg) and a high dose group (200 mg/kg), a 50-100kDa roughhaired holly root polysaccharide low dose group and a high dose group, and a roughhaired holly root polysaccharide low dose group and a high dose group with more than 100kDa roughhaired holly root polysaccharide;
(3) The mice are molded by cyclophosphamide for 5 days, after the molding is successful, roughhaired holly root polysaccharide treatment is carried out for 7 days, and all mice are weighed every day for fixed time and weight changes are recorded;
(4) Blood was collected by eyeball into sterile tube, centrifuged at 3000r/min and 4℃for 10min to extract supernatant serum and assayed for TNF- α and IL-6 by experimental ELISA kit.
(5) Spleens of the rapidly dissected mice were rinsed with PBS solution, stored in 4% paraformaldehyde for 24h, processed and embedded in paraffin. Sections 5 μm thick were prepared and stained with hematoxylin and eosin. Spleen histology and morphology were observed using a microscope.
As shown in fig. 5, the experimental results of weight change show that the weight of each group of mice except the control group was decreased in the former 5-day cyclophosphamide modeling period, and that the weight of the mice was increased and dose-dependent in the 6-day treatment group, and the increase rate was as follows: IAPS1-H > IAPS1-L > IAPS2-H > IAPS3-H > IAPS2-L > IAPS3-L > CTX, and the increase rate of the treatment group is higher than that of the control group, and the result shows that the roughhaired holly root polysaccharide has an immunoregulatory effect and the IAPS1 has the best effect.
The spleen index can intuitively reflect the immunity of the mice. Spleen index= (spleen weight (mg))/(body weight (g)), the result of which is shown in fig. 5, the spleen index of the model group is significantly reduced compared to the blank group; compared with the model group, the IAPS group treated by the roughhaired holly root polysaccharide with different molecular weights can obviously improve the spleen index of the immunosuppressed mice, and the IAPS1 effect is optimal at a high dose.
TNF- α is a pro-inflammatory cytokine that not only promotes macrophage activation and antigen presentation, but also plays an increasing role in mediating innate and adaptive immune responses. The results for TNF- α are shown in FIG. 6, where the different molecular weight roughhaired holly root polysaccharides significantly increased TNF- α production in a concentration-dependent manner compared to the control group, and IAPS1 had significantly higher effect on TNF- α than IAPS2 and IAPS3. And the content is IAPS1-H > IAPS1-L > IAPS2-H > IAPS2-L > IAP3S-H > IAPS3-L sequentially from high to low. This suggests that high doses of roughhaired holly root polysaccharide can improve the inflammatory infiltrate induced by CTX, with a stronger immune effect.
IL-6 cytokine production is an important pathway for evaluation of macrophage immunomodulation, and the results are shown in FIG. 7. It can be seen that the different molecular weight roughhaired holly root polysaccharides significantly increased IL-6 production in a concentration dependent manner compared to the control group, and that IAPS1 had significantly higher effect on IL-6 than IAPS2 and IAPS3. The content is IAPS1-H > IAPS1-L > IAPS2-H > IAPS2-L > IAP3S-H > IAPS3-L sequentially from high to low, which shows that IAPS1-H has the best effect of relieving inflammation caused by CTX model.
The effect of roughhaired holly root polysaccharide 1-3 on CTX-induced immunosuppression mouse model was observed by spleen histology, and the results are shown in FIGS. 9-11, in which the black boxes are partially enlarged, and the black arrows represent megakaryocyte precursor cells.
As can be seen from the figure, the spleen of the control group has clear structure, and spleen nodules consisting of closely arranged lymphocytes and red marrow surrounding the spleen nodules containing a large number of red blood cells are obvious in red and white marrow limitation; compared with a blank group, the model group has the advantages that the cell number is reduced, the structure is fuzzy, the boundary between the red marrow and the edge area is not obvious, and the multinuclear giant cells are increased;
the high dose groups IAPS1, IAPS2 and IAPS3 can significantly increase the cell number in the spleen marginal zone of the immunosuppressed mice and increase the cell number, and the red and white marrow limit is obvious.
In the lymphoid tissue, the cells of the control group are orderly arranged, compared with the control group, the multinuclear giant cells of the model group are obviously increased, the CTX-induced immunosuppression model can be relieved through the treatment of roughhaired holly root polysaccharide 1-3, and the pathological phenomena of the model by high-dose IAPS1, IAPS2 and IAPS3 are obviously improved. The test results show that different roughhaired holly root polysaccharides 1-3 can antagonize immune organ injury caused by CTX to a certain extent, and the higher the concentration, the better the improvement effect, which is beneficial to maintaining the normal structure and function of immune organs.
In conclusion, the roughhaired holly root polysaccharide prepared by the invention can effectively inhibit the enhancement of tumor cells, wherein the inhibition effect on lung cancer cells is optimal;
second, roughhaired holly root polysaccharide has an inhibitory effect on macrophage iron death caused by inflammation, and thus can provide immunity through macrophage activity.
Second, roughhaired holly root polysaccharide can also reduce inflammation by modulating inflammatory factors.
Claims (1)
1. The application of roughhaired holly root polysaccharide in preparing tumor cell proliferation inhibition drugs is characterized in that roughhaired holly root polysaccharide is roughhaired holly root polysaccharide 1, and monosaccharide composition of roughhaired holly root polysaccharide 1 is 1.04% rha,0.70% ara,3.05% Gal,78.98% Glc,1.29% Man,4.65% Gal-UA,5.80% Gul-UA,1.28% Glc-UA and 3.22% Man-UA;
the tumor cells are lung cancer cells, liver cancer cells or colorectal cancer cells.
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