CN109251253A - A kind of Rubus chingii polysaccharide and preparation method thereof and preparing the application in liver cell Fatty toxicity damage inhibitors - Google Patents
A kind of Rubus chingii polysaccharide and preparation method thereof and preparing the application in liver cell Fatty toxicity damage inhibitors Download PDFInfo
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Abstract
The invention discloses a kind of Rubus chingii polysaccharide and its preparation process and applications, the Rubus chingii polysaccharide is mentioned by ultrasonic wave added water to be prepared with alcohol precipitation process, with molar percent, which includes following monosaccharide: galacturonic acid 40.26~45.20%；Arabinose 29.78~33.17%；Galactolipin 7.63~9.59%；Glucose 6.38~9.65%；Mannose 1.18~1.31%；Rhamnose 3.29~4.89%；Glucuronic acid 0.98~1.13%；Fucose 0.27~0.65%.The Rubus chingii polysaccharide can be obviously improved the survival rate of the liver cell of palmitinic acid induced damage, significant decrease palmitinic acid induces the cell ROS generated is horizontal, palmitinic acid is effectively relieved to induce mitochondrial membrane potential in anoxic level reduction of generation and other effects, therefore, it can be used for preparing liver cell Fatty toxicity damage inhibitors.
Description
Technical field
The present invention relates to the technical fields of plant polyose, and in particular to a kind of Rubus chingii polysaccharide and preparation method thereof and
Preparing the application in liver cell Fatty toxicity damage inhibitors.
Background technique
Rubus chingii (latin name: Rubus chingii Hu), also known as male, milk mother of rubus chingii Hu, big bugle etc.,
Rose family rubus plant is distributed in the ground such as Jiangsu, Anhui, Zhejiang, Jiangxi, Fujian,.Immature fruit can be used as medicine, " China
Pharmacopeia " record its slightly warm in nature, it is sweet in flavor, sour, enter liver kidney channel, tool nourishing liver and kidney, reduce just, supporing yang controlling nocturnal emission with astringent drugs, the effect of improving eyesight, cure mainly sun
The diseases such as impotence, spermatorrhea, consumptive disease, mesh be dark.Ripening fruits delicate mouthfeel, sweet and sour taste is full of nutrition, is rich in amino acid, vitamin
C, the ingredients such as vitamin E and vitamin B3, nutritive value and medical care effect with higher.Modern research shows that it is led
Ingredient is wanted to have terpene, flavones etc., pharmacological action mainly has antimutagenesis, improving studing ability, anti-aging, moreover it is possible to increase
It is strong immune, there is (the Jiangxi progress [J] the Chinese medicine of the peaceful raspberry of Miao Juru, Xie Yihui, Liu Hong such as regulating and controlling effect to gonad axis
Medicine, 2004,35 (1): 54-55.).In addition, Liu Mingxue etc. (Liu Mingxue, Niu Jinge, Su Zhongwei wait raspberry Polyose extraction,
Structural analysis and radicals scavenging Effect study [J] food science and technology, 2009 (7): 163-167.) it is sent out using in-vitro evaluation system
Existing, raspberry polysaccharide has stronger Scavenging activity to oxygen radical, however does not have correlative study in cell and internal level；Its
During purified polysaccharide, using the methods of NaOH and CTAB solution precipitate polysaccharides, step is complicated；Simultaneously because being purified using alkaline process,
The structure of destructible polysaccharide, polysaccharide loss are larger.
Polysaccharide (polysaccharide) is the sugar combined by glycosidic bond formed by the condensation of multiple monosaccharide molecules, dehydration
Chain is that structure is complicated and huge polymeric carbohydrate for molecule.Polysaccharide composition is sufficiently complex with structure, is not a kind of
Pure chemical substance, but the mixture of the different substance of extent of polymerization.Polysaccharide is a kind of life with extensive bioactivity
Object macromolecular has a variety of pharmacological activity, such as anti-oxidant, hypoglycemic, reducing blood lipid, antitumor and strengthen immunity, has
Higher Development volue.As Li Guilan (extraction of Li Gui orchid raspberry polysaccharide and products thereof develop [D] University Of Nanchang,
2014.) technique that inner ebullition method extracts raspberry polysaccharide is established, 80 DEG C of Extracting temperature of discovery, extracts liquid-to-solid ratio 10:1
Raspberry polysaccharide yield highest when time 10min.One is disclosed in the Chinese patent literature of 107266603 A of Publication No. CN
Kind has the raspberry polysaccharide component extracting method for removing free radical function, passes through polyamide column and DEAE-cellulose yin
Ion column purified polysaccharide, however the step of passing through column purification is many and diverse, and the prices of raw materials are expensive.(Xu Liping, the Wu Yuan such as Xu Liping
Beautiful woman, Wang Xin wait Response Surface Method to optimize raspberry polysaccharide extracting process [J] Food Additives Used in China, 2017 (9): 182-
187.) using response phase method research solid-liquid ratio, Extracting temperature, extraction time to raspberry (rubus) Polyose extraction yield
It influences, the raspberry polysaccharide that the method obtains is mixture, contains the impurity such as polyphenol, albumen.Currently, not yet discovery high-purity is slapped
The rapid extracting method of leaf raspberry polysaccharide.
Liver is the vitals of human-body biological metabolism and removing toxic substances, and saturated fatty acid content is excessively high to cause hepar damnification.
Palmitinic acid (Palmitic acid, PA) is a kind of free fatty acid that content is most in blood of human body, while being also free-fat
Most important saturated fatty acid in acid.Palmitinic acid is widely used in the in vitro studies such as Fatty toxicity and metabolic syndrome.Therefore, inhibit palm fibre
Palmitic acid acid has very big research significance to the substance that liver Fatty toxicity damages.
Summary of the invention
Based on above-mentioned technical problem, the present invention is mentioned by ultrasonic wave added water and a kind of composition spy is prepared with alcohol precipitation process
Very, the Rubus chingii polysaccharide of high-purity, the Rubus chingii polysaccharide can be obviously improved the liver cell of palmitinic acid induced damage
Survival rate, significantly reduce that the cell ROS that palmitinic acid induction generates is horizontal, the cell line that palmitinic acid induction generates is effectively relieved
The cell GSH that mitochondrial membrane potential level reduced, effectively reduced palmitinic acid induction generation is consumed and is effectively restored palmitinic acid induction and produces
Therefore raw cell nuclear damage can be used for preparing liver cell Fatty toxicity damage inhibitors.
Specific technical solution is as follows:
A kind of Rubus chingii polysaccharide, with molar percent, the Rubus chingii polysaccharide includes following monosaccharide:
The constitutional repeating unit that polysaccharide is made of monosaccharide with certain connection type, monosaccharide are connected with certain glycosidic bond
It connects, the monosaccharide of the connection of different glycosidic bonds is known as the structural units of polysaccharide.With molar ratio computing, the Rubus chingii polysaccharide includes
Following structural units:
Preferably, the weight average molecular weight of the Rubus chingii polysaccharide is 758.03~837.82kDa.
The invention also discloses the preparation methods of the Rubus chingii polysaccharide, including following extraction step:
(1) Rubus chingii crushed, clean after obtain raw material powder；
(2) raw material powder is after ultrasonic wave added water mentions, then sediment is collected after alcohol precipitation, is denoted as crude extract；
(3) crude extract is soluble in water, proteinaceous impurities are removed, the water phase of collection is filtered through bag filter to be retained, filter
Liquid obtains the Rubus chingii polysaccharide again through vacuum freeze drying out.
The present invention is mentioned using ultrasonic wave added water and alcohol precipitation process, is realized by the accuracy controlling to technological parameter and is covered to palm leaf
The Effective Regulation of the content of each monosaccharide and each structural units in basin polysaccharide.
In step (1), after the Rubus chingii freeze-drying, is crushed through micronizer, cross 40 meshes, reuse anhydrous second
Alcohol extracts the oil-soluble impurities in Rubus chingii, collects precipitating, as raw material powder.
In step (2), the ultrasonic wave added water is mentioned, and ultrasonic power is 150~250W, and Extracting temperature is 70~95 DEG C, when
Between be 2~4h, number of repetition be 3~6 times.
The ethyl alcohol that volumetric concentration is 70~95% is added in the alcohol precipitation, unless otherwise specified, certain density in the present invention
Ethyl alcohol is ethanol-water solution.Alcohol precipitation number is 3~5 times, because containing a large amount of oil-soluble impurities in Rubus chingii, is being mentioned
It can be removed by this step during taking polysaccharide.
In step (3):
Proteinaceous impurities are removed using Sevag method, number of repetition is 6~9 times, can effectively be removed in Rubus chingii polysaccharide
Proteinaceous impurities.
Bag filter retention can remove the small molecular weight impurity in Rubus chingii polysaccharide, obtain high-purity Rubus chingii
Polysaccharide；Preferably, the bag filter filtering molecular cut off is greater than the ingredient of 3.5kDa.
It is further preferred:
The Extracting temperature that the ultrasonic wave added water mentions is 80~95 DEG C, and the alcohol precipitation uses volumetric concentration for 70~90%
Ethyl alcohol.
It is found through experiment that the Rubus chingii polysaccharide extracted using above-mentioned further preferred process conditions, to rub
That percentages, including following monosaccharide:
With molar ratio computing, the Rubus chingii polysaccharide includes following structural units:
A series of activity test discovery has been carried out for the Rubus chingii polysaccharide that above-mentioned special process extracts:
Extracting obtained Rubus chingii polysaccharide has protective effect to the liver cell Fatty toxicity damage that palmitinic acid induces,
It is embodied in:
1, the survival rate of the liver cell of palmitinic acid induced damage can be obviously improved；
2, it is horizontal to significantly reduce the cell ROS that palmitinic acid induction generates；
3, it is horizontal that the cellular superoxide anion that palmitinic acid induction generates is effectively reduced；
4, the mitochondrial membrane potential in anoxic level that palmitinic acid induction generates, which is effectively relieved, to be reduced；
5, the cell GSH consumption that palmitinic acid induction generates is effectively reduced；
6, effectively restore the cell nuclear damage that palmitinic acid induction generates.
Therefore, the invention also discloses the Rubus chingii polysaccharide in preparing liver cell Fatty toxicity damage inhibitors
Using.Specifically, the liver cell Fatty toxicity through palmitinic acid induction damages.
Preferably, in the inhibitor, the effective dose of Rubus chingii polysaccharide is 150 μ g/mL.
Further preferably, with molar percent, the Rubus chingii polysaccharide includes following monosaccharide:
With molar ratio computing, the Rubus chingii polysaccharide includes following structural units:
It is found through activity test, the Rubus chingii polysaccharide of above-mentioned composition is for the liver generated by palmitinic acid induction
There is more significant inhibiting effect in cytolipin toxic damages.
Compared with prior art, the present invention has the advantage that
1, the present invention is mentioned and alcohol precipitation process using ultrasonic wave added water, and being obtained by the accuracy controlling to technological parameter has spy
It is fixed at being grouped as, the Rubus chingii polysaccharide of molecular weight and glycosidic bond.
2, present invention firstly discovers that the Fatty toxicity damage that Rubus chingii polysaccharide can effectively inhibit palmitinic acid to induce, is
Rubus chingii polysaccharide provides new medical application, has expanded a new application field.
Detailed description of the invention
Fig. 1 is that the Rubus chingii polysaccharide that Examples 1 to 4 is prepared respectively aoxidizes the liver cell LO2 that palmitinic acid induces
The protective effect of damage；
Fig. 2 is the liver cell LO2 activity that the Rubus chingii polysaccharide that Examples 1 to 4 is prepared respectively induces palmitinic acid
The inhibiting effect of oxygen radical ROS outburst；
Fig. 3 is the liver cell LO2 super oxygen that the Rubus chingii polysaccharide that Examples 1 to 4 is prepared respectively induces palmitinic acid
The inhibiting effect of compound anion；
Fig. 4 is the liver cell LO2 line grain that the Rubus chingii polysaccharide that Examples 1 to 4 is prepared respectively induces palmitinic acid
The relaxation effect that body level of membrane potential reduces；
Fig. 5 is the liver cell LO2GSH that the Rubus chingii polysaccharide that Examples 1 to 4 is prepared respectively induces palmitinic acid
The relaxation effect of consumption；
Fig. 6 is the liver cell LO2 nuclear damage that lotus Rubus chingii polysaccharide prepared by embodiment 1 induces palmitinic acid
Inhibiting effect.
Specific embodiment
Present invention is further described in detail with reference to the accompanying drawings and embodiments, it should be pointed out that following embodiment
It is intended to convenient for the understanding of the present invention, and does not play any restriction effect to it.
Embodiment 1
It after Rubus chingii freeze-drying, is crushed through micronizer, crosses 40 meshes, extract Rubus chingii using dehydrated alcohol
In liposoluble substance, be repeated 3 times, collect precipitating；Sediment further uses ultrasonic wave added hot water to extract, ultrasonic power
250W, Extracting temperature are 90 DEG C, and extraction time 2h is repeated 4 times；Ethyl alcohol is added in extracting solution, keeps ethyl alcohol volume in system dense
Degree is 70%, collects precipitating.It will precipitate soluble in water, using Sevag method removing protein class impurity, be repeated 7 times, it is molten to collect water phase
Liquid.Solution is greater than the component of 3.5kDa through bag filter filtering molecular cut off, and filter liquor through vacuum freeze drying, obtains palm leaf again
Raspberry polysaccharide.
Rubus chingii polysaccharide manufactured in the present embodiment, weight average molecular weight 758.03kDa.It is tested through liquid chromatogram
It is found that the main component of the Rubus chingii polysaccharide is as shown in table 1 below:
Table 1
| Ingredient | Molar percentage |
| Galacturonic acid | 45.20% |
| Arabinose | 32.65% |
| Galactolipin | 8.53% |
| Glucose | 7.63% |
| Mannose | 1.19% |
| Rhamnose | 3.37% |
| Glucuronic acid | 1.05% |
| Fucose | 0.38% |
Through the measuring that methylates, with molar ratio computing, Rubus chingii polysaccharide manufactured in the present embodiment is by specific in table 2
Structural units's composition:
Table 2
Embodiment 2
It after Rubus chingii freeze-drying, is crushed through micronizer, crosses 40 meshes, extract Rubus chingii using dehydrated alcohol
In liposoluble substance, be repeated 4 times, collect precipitating；Sediment further uses ultrasonic wave added hot water to extract, ultrasonic power
200W, Extracting temperature are 80 DEG C, and extraction time 3h is repeated 6 times；Ethyl alcohol is added in extracting solution, keeps ethyl alcohol volume in system dense
Degree is 80%, collects precipitating.It will precipitate soluble in water, using Sevag method removing protein class impurity, be repeated 9 times, it is molten to collect water phase
Liquid.Solution is greater than the component of 3.5kDa through bag filter filtering molecular cut off, and filter liquor through vacuum freeze drying, obtains palm leaf again
Raspberry polysaccharide.
Rubus chingii polysaccharide manufactured in the present embodiment, weight average molecular weight 837.82kDa.It is tested through liquid chromatogram
It is found that the main component of the Rubus chingii polysaccharide is as shown in table 3 below:
Table 3
| Ingredient | Molar percentage |
| Galacturonic acid | 44.21% |
| Arabinose | 32.17% |
| Galactolipin | 9.57% |
| Glucose | 7.54% |
| Mannose | 1.26% |
| Rhamnose | 3.54% |
| Glucuronic acid | 1.13% |
| Fucose | 0.58% |
Through the measuring that methylates, with molar ratio computing, the Rubus chingii polysaccharide of the present embodiment is by following specific structure list
Position composition:
Table 4
| Ingredient | Molar ratio |
| 1,3- arabinose | 2.69 |
| 1,6- galactolipin | 2.24 |
| T- galacturonic acid | 1.86 |
| 1,6- glucose | 1.25 |
| 1,4- galacturonic acid | 1.17 |
| 1,4,6- galactolipin | 1.21 |
| 1,4- rhamnose | 1.00 |
| 1,2- arabinose | 0.80 |
| 1,4- arabinose | 0.67 |
| T- arabinose | 0.36 |
| T- glucuronic acid | 0.30 |
| 1,3- fucose | 0.29 |
| 1,3- mannose | 0.28 |
| 1,4- glucuronic acid | 0.24 |
Embodiment 3
It after Rubus chingii freeze-drying, is crushed through micronizer, crosses 40 meshes, extract Rubus chingii using dehydrated alcohol
In liposoluble substance, be repeated 5 times, collect precipitating；Sediment further uses ultrasonic wave added hot water to extract, ultrasonic power
150W, Extracting temperature are 95 DEG C, and extraction time 2h is repeated 3 times；Ethyl alcohol is added in extracting solution, keeps ethyl alcohol volume in system dense
Degree is 90%, collects precipitating.It will precipitate soluble in water, using Sevag method removing protein class impurity, be repeated 6 times, it is molten to collect water phase
Liquid.Solution is greater than the component of 3.5kDa through bag filter filtering molecular cut off, and filter liquor through vacuum freeze drying, obtains palm leaf again
Raspberry polysaccharide.
Rubus chingii polysaccharide manufactured in the present embodiment, weight average molecular weight 823.68kDa.It is tested through liquid chromatogram
It is found that the main component of the Rubus chingii polysaccharide is as shown in table 5 below:
Table 5
| Ingredient | Molar percentage |
| Galacturonic acid | 44.44% |
| Arabinose | 31.71% |
| Galactolipin | 9.15% |
| Glucose | 6.95% |
| Mannose | 1.29% |
| Rhamnose | 4.89% |
| Glucuronic acid | 1.09% |
| Fucose | 0.48% |
Through the measuring that methylates, with molar ratio computing, the Rubus chingii polysaccharide of the present embodiment is by following specific structure list
Position composition:
Table 6
| Ingredient | Molar ratio |
| 1,3- arabinose | 2.49 |
| 1,6- galactolipin | 2.22 |
| T- galacturonic acid | 2.01 |
| 1,6- glucose | 1.17 |
| 1,4- galacturonic acid | 1.26 |
| 1,4,6- galactolipin | 1.03 |
| 1,4- rhamnose | 0.75 |
| 1,2- arabinose | 0.79 |
| 1,4- arabinose | 0.58 |
| T- arabinose | 0.46 |
| T- glucuronic acid | 0.30 |
| 1,3- fucose | 0.29 |
| 1,3- mannose | 0.18 |
| 1,4- glucuronic acid | 0.24 |
Embodiment 4
It after Rubus chingii freeze-drying, is crushed through micronizer, crosses 40 meshes, extract Rubus chingii using dehydrated alcohol
In liposoluble substance, be repeated 5 times, collect precipitating；Sediment further uses ultrasonic wave added hot water to extract, ultrasonic power
220W, Extracting temperature are 70 DEG C, and extraction time 4h is repeated 6 times；Ethyl alcohol is added in extracting solution, keeps ethyl alcohol volume in system dense
Degree is 95%, collects precipitating.It will precipitate soluble in water, using Sevag method removing protein class impurity, be repeated 8 times, it is molten to collect water phase
Liquid.Solution is greater than the component of 3.5kDa through bag filter filtering molecular cut off, and filter liquor through vacuum freeze drying, obtains palm leaf again
Raspberry polysaccharide.
Rubus chingii polysaccharide manufactured in the present embodiment, weight average molecular weight 787.66kDa.It is tested through liquid chromatogram
It is found that the main component of the Rubus chingii polysaccharide is as shown in table 7 below:
Table 7
| Ingredient | Molar percentage |
| Galacturonic acid | 43.62% |
| Arabinose | 30.97% |
| Galactolipin | 8.84% |
| Glucose | 9.55% |
| Mannose | 1.29% |
| Rhamnose | 3.99% |
| Glucuronic acid | 1.11% |
| Fucose | 0.63% |
Through the measuring that methylates, with molar ratio computing, the Rubus chingii polysaccharide of the present embodiment is by following specific structure list
Position composition:
Table 8
Performance test: research Rubus chingii polysaccharide makees the protection for the liver cell Fatty toxicity damage that palmitinic acid induces
With the Rubus chingii polysaccharide prepared respectively with Examples 1 to 4 is tested (1) cell survival rate measurement (mtt assay)
According to the method for bibliography, using the survival rate of mtt assay detection cell.The LO2 cell inoculation of logarithmic phase is arrived
(concentration is 4.5 × 10 in 96 porocyte culture plates3A cells/well), after being incubated for for 24 hours；In the item containing Rubus chingii polysaccharide
Under part, for 24 hours with palmitinic acid (PA 0.1mM) processing LO2 cell, 4h then is incubated for MTT (0.5mg/mL).The sediment of generation
It is dissolved in the DMSO of 200 μ L, with the absorbance at microplate reader check fee 490nm.
Cell survival rate (%)=[ASample/ABlank] × 100%；
If Fig. 1 is shown, LO2 cell is after 0.1mM PA is damaged for 24 hours, and cell density is substantially reduced, compared with normal group, carefully
Born of the same parents' survival rate is 52.96%, is had statistical significance (p < 0.05).Rubus chingii polysaccharide is under the effect of 0.1mM PA concentration
When, there is preferable protective effect to LO2 cell, compared with the control group, there is significant difference, 150 μ g/mL Rubus chingiis are more
Under sugared (embodiment 1) effect, cell survival rate is up to 75.00% (p < 0.05), and the function and effect of Examples 1 to 3 are substantially better than
Embodiment 4.
Above results demonstrate that Rubus chingii polysaccharide has the protection for the cytolipin toxic damages for intervening palmitinic acid induction
Effect.
(2) intracellular ROS level detects
Collect logarithmic phase LO2 cell, seed cells into 24 porocyte culture plates (concentration be 3.5 × 104It is a thin
Born of the same parents/hole), after being incubated for for 24 hours；Under conditions of containing Rubus chingii polysaccharide, LO2 cell is handled with palmitinic acid (PA 0.1mM)
For 24 hours, it is then dyed with DCFH-DA, 37 DEG C of incubation 30min, with fluorescence microscope and takes pictures after dyestuff is cleaned, then divide
Analyse its fluorescence intensity.
If Fig. 2 is shown, compared with untreated control group (control), for 24 hours through 0.1mM PA effect liver cell LO2,
(fluorescence intensity 137.72%) is remarkably reinforced in fluorescence intensity.Rubus chingii polysaccharide can significantly reduce what palmitinic acid induction generated
ROS fluorescence intensity, and there is statistical significance, 150 μ g/mL Rubus chingii polysaccharide (embodiment 1) effects are best, and (fluorescence is strong
It spends 102.8%), and the function and effect of Examples 1 to 3 are substantially better than embodiment 4.
It is horizontal that the above results show that the cell ROS that PA induction generates can be effectively reduced in Rubus chingii polysaccharide.
(3) intracellular superoxide anion level detects
Collect logarithmic phase LO2 cell, seed cells into 24 porocyte culture plates (concentration be 3.5 × 104It is a thin
Born of the same parents/hole), after being incubated for for 24 hours；Under conditions of containing Rubus chingii polysaccharide, LO2 cell is handled with palmitinic acid (PA 0.1mM)
For 24 hours, it is then dyed with DHE, 37 DEG C of incubation 30min, with fluorescence microscope and takes pictures after dyestuff is cleaned, then analyze
Its fluorescence intensity.
If Fig. 3 is shown, compared with untreated control group (control), for 24 hours through 0.1mM PA effect liver cell LO2,
(fluorescence intensity 151.30%) is remarkably reinforced in fluorescence intensity.Rubus chingii polysaccharide can significantly reduce what palmitinic acid induction generated
ROS fluorescence intensity, and there is statistical significance, 150 μ g/mL Rubus chingii polysaccharide (embodiment 2) effects are best, and (fluorescence is strong
It spends 99.12%), and the function and effect of Examples 1 to 3 are substantially better than embodiment 4.
The above results show that the cellular superoxide anionic water that PA induction generates can be effectively reduced in Rubus chingii polysaccharide
It is flat.
(4) intracellular mitochondrial level of membrane potential detects
Collect logarithmic phase LO2 cell, seed cells into 24 porocyte culture plates (concentration be 3.5 × 104It is a thin
Born of the same parents/hole), after being incubated for for 24 hours；Under conditions of containing Rubus chingii polysaccharide, LO2 cell is handled with palmitinic acid (PA 0.1mM)
For 24 hours, it is then dyed with Rh123,37 DEG C of incubation 30min, with fluorescence microscope and takes pictures after dyestuff is cleaned, then analyze
Its fluorescence intensity.
If Fig. 4 is shown, compared with untreated control group (control), for 24 hours through 0.1mM PA effect liver cell LO2,
Fluorescence intensity is substantially reduced (fluorescence intensity 83.65%).Rubus chingii polysaccharide can significantly alleviate the line that palmitinic acid induction generates
Mitochondrial membrane potential fluorescence intensity reduces, and has statistical significance, and 150 μ g/mL Rubus chingii polysaccharide (embodiment 1) effects are most
Good (fluorescence intensity 101.90%).
It is horizontal that the above results show that the mitochondrial membrane potential in anoxic that PA induction generates can be effectively relieved in Rubus chingii polysaccharide
It reduces.
(5) intracellular GSH level detection
Collect logarithmic phase LO2 cell, seed cells into 24 porocyte culture plates (concentration be 3.5 × 104It is a thin
Born of the same parents/hole), after being incubated for for 24 hours；Under conditions of containing Rubus chingii polysaccharide, LO2 cell is handled with palmitinic acid (PA 0.1mM)
For 24 hours, it is then dyed with NDA, 37 DEG C of incubation 30min, with fluorescence microscope and takes pictures after dyestuff is cleaned, then analyze
Its fluorescence intensity.
If Fig. 5 is shown, compared with untreated control group (control), for 24 hours through 0.1mM PA effect liver cell LO2,
(fluorescence intensity 50.00%) is remarkably reinforced in fluorescence intensity.Rubus chingii polysaccharide can significantly alleviate the GSH that palmitinic acid induction generates
Fluorescence intensity reduces, and has statistical significance, and 150 μ g/mL Rubus chingii polysaccharide (embodiment 1) effects are best, and (fluorescence is strong
It spends 74.85%), and the function and effect of Examples 1 to 3 are substantially better than embodiment 4.
The above results show that Rubus chingii polysaccharide can effectively reduce the cell GSH consumption that PA induction generates.
(6) cell nuclear damage Hochest level detects
Collect logarithmic phase LO2 cell, seed cells into 24 porocyte culture plates (concentration be 4.0 × 104It is a thin
Born of the same parents/hole), after being incubated for for 24 hours；Under conditions of containing Rubus chingii polysaccharide, LO2 cell is handled with palmitinic acid (PA 0.1mM)
For 24 hours, PBS is dyed after cleaning with fluorescence probe, 37 DEG C of incubation 10-15min, after dyestuff is cleaned simultaneously with fluorescence microscope
It takes pictures, then analysis picture.
If Fig. 6 is shown, normal LO2 cell (control) nucleus volume is larger, chromatin be it is uniform, through palmitinic acid
(PA 0.1mM) acts on LO2 cell, and the chromatin of part cell is obviously concentrated, and apoptotic body occurs, and is added 150
After μ g/mL Rubus chingii polysaccharide (embodiment 1) pretreatment for 24 hours, chromatin is slightly unfolded, and karyomorphism is restored.
The above results show that Rubus chingii polysaccharide can effectively restore the cell nuclear damage that PA induction generates.
Finally, the present invention can be summarized with others without prejudice to the concrete form of spirit or central characteristics of the invention.Cause
This, no matter from the point of view of that point, the embodiment above of the invention can only all be considered the description of the invention and cannot limit
The present invention, claims indicate the scope of the present invention, and above-mentioned explanation does not point out the scope of the present invention, therefore,
With any change in the comparable meaning and scope of claims of the present invention, all it is considered as including in claims
In range.
Claims (10)
1. a kind of Rubus chingii polysaccharide, which is characterized in that with molar percent, the Rubus chingii polysaccharide includes following
Monosaccharide:
2. Rubus chingii polysaccharide according to claim 1, which is characterized in that with molar ratio computing, the Rubus chingii
Polysaccharide includes following structural units:
3. Rubus chingii polysaccharide according to claim 1 or 2, which is characterized in that weight average molecular weight be 758.03~
837.82kDa。
4. a kind of preparation method of the Rubus chingii polysaccharide any according to claim 1~3, which is characterized in that including
Following extraction step:
(1) Rubus chingii crushed, clean after obtain raw material powder；
(2) raw material powder is after ultrasonic wave added water mentions, then sediment is collected after alcohol precipitation, is denoted as crude extract；
(3) crude extract is soluble in water, proteinaceous impurities are removed, the water phase of collection is filtered through bag filter to be retained, filter liquor
Again through vacuum freeze drying, the Rubus chingii polysaccharide is obtained.
5. the preparation method of Rubus chingii polysaccharide according to claim 4, which is characterized in that described super in step (2)
Sound auxiliary water mentions, and ultrasonic power is 150~250W, and Extracting temperature is 70~95 DEG C.
6. the preparation method of Rubus chingii polysaccharide according to claim 4, which is characterized in that in step (2), the alcohol
It is heavy, the ethyl alcohol that volumetric concentration is 70~95% is added.
7. the preparation method of Rubus chingii polysaccharide according to claim 4, which is characterized in that in step (3):
Proteinaceous impurities are removed using Sevag method, number of repetition is 6~9 times；
The bag filter filtering molecular cut off is greater than the ingredient of 3.5kDa.
8. according to the preparation method of any Rubus chingii polysaccharide of claim 4~7, which is characterized in that the ultrasound
The Extracting temperature that auxiliary water mentions be 80~95 DEG C, the alcohol precipitation use volumetric concentration for 70~90% ethyl alcohol.
9. a kind of Rubus chingii polysaccharide any according to claim 1~3 is preparing the damage suppression of liver cell Fatty toxicity
Application in preparation.
10. application according to claim 9, which is characterized in that with molar percent, the Rubus chingii polysaccharide packet
Include following monosaccharide:
With molar ratio computing, the Rubus chingii polysaccharide includes following structural units:
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| CN115960274A (en) * | 2022-12-23 | 2023-04-14 | 温州医科大学 | Raspberry polysaccharide and preparation method and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114917246A (en) * | 2022-05-30 | 2022-08-19 | 华南师范大学 | Application of raspberry polysaccharide R2 in preparing antitumor drugs and anti-inflammatory preparations |
| CN114917245A (en) * | 2022-05-30 | 2022-08-19 | 华南师范大学 | Application of raspberry polysaccharide R1 in preparing antitumor drugs and anti-inflammatory preparations |
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| CN115960274A (en) * | 2022-12-23 | 2023-04-14 | 温州医科大学 | Raspberry polysaccharide and preparation method and application thereof |
| CN115960274B (en) * | 2022-12-23 | 2024-04-09 | 温州医科大学 | Raspberry polysaccharide and preparation method and application thereof |
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