CN103550243A - Application of atractylodes macrocephalaon polysaccharide in medicaments or healthcare products for improving kuffer cell immune function - Google Patents
Application of atractylodes macrocephalaon polysaccharide in medicaments or healthcare products for improving kuffer cell immune function Download PDFInfo
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Abstract
The invention relates to application of atractylodes macrocephalaon polysaccharide in medicaments or healthcare products for improving kuffer cell immune function, belonging to the technical field of traditional Chinese medicine. According to the application, homemade PAM (atractylodes macrocephalaon polysaccharide) is adopted for testing the influences on the mouse Kupffer cell immune function. The PAM in vitro is acted to a normal Kupffer cell so as to evaluate the immune function and the cell damage situation according to the phagocytosis quantity of neutral red A540, the secreting quantity of NO and TNF (tumor necrosis factor)-alpha as well as the LDH (lactate dehydrogenase) leakage quantity; after in-vivo mice are injected with the PAM, the indexes of the Kupffer cell, the activity of intracellular ACP (acid phosphatase) as well as the serum ALT and GST are detected. The result shows that the PAM intensifies the phagocytosis to the neutral red caused by Kupffer cell, improves the ACP activity as well as increases the generation amount of NO and TNF-alpha. The PAM in vitro does not increase parenchymal hepatic cell LDH leakage, and has no influence on the activities of sALT and sGST, so that the PAM can activate the Kupffer cell immune function, improves the organism disease resistance, and has an actual application value at the treatment aspect of diseases of immunodeficiency disease, tumor and the like.
Description
Technical field
The present invention relates to technical field of Chinese medicines, particularly relate to a kind of Chinese medicine extract Rhizoma Atractylodis polysaccharide and improving the medicine of kuffer cellular immune function or the application in health product.
Background technology
In medicinal plants, exist immunocompetence polysaccharide widely, it has inhibition tumor growth, and immune cell activated improves the several functions such as body's immunity.The Rhizoma Atractylodis Macrocephalae is the dry rhizome of feverfew Rhizoma Atractylodis Macrocephalae Atractylodes macrophala Koedz, is one of famous " Zhejiang eight tastes ".The Rhizoma Atractylodis Macrocephalae is the most frequently used traditional tonic medicine, has " Nan Shubei ginseng " laudatory title, has invigorating the spleen and benefiting QI, stomach reinforcing, dampness diuretic, and in, hidroschesis, antiabortive effect.Rhizoma Atractylodis polysaccharide (PAM) is the water soluble polysaccharide extracting in Rhizoma Atractylodis Macrocephalae plant material, and its main component is mannan and levan, is the important activity composition that the Rhizoma Atractylodis Macrocephalae is brought into play its immunologic enhancement." progress of Rhizoma Atractylodis polysaccharide immunoregulation effect " (herding and veterinary, 2010,42 volumes, 98-100 page) summarized the regulating action of PAM to humoral immunization, regulating action to cellular immunization, the regulation and control to cytokine, mainly concentrate on PAM and can obviously improve B, T lymphopoiesis.Kuffer cell is important immunologically competent cell in body, accounts for 90% and secretion various kinds of cell of body tissue macrophage phagocytic ability because and the function of hepatic parenchymal cells is had to certain regulating action.How PAM affects Kuffer cell function, there is not yet report.
Summary of the invention
Blank for above-mentioned field, the present invention adopts external and vivo experiment method, observe the impact of PAM on normal rat Kuffer cell function, show that PAM can activate Kupffer cellular immune function, improve body resistance against diseases, aspect the treatment of the diseases such as immunodeficiency and tumor, there is actual application value.
The present invention simultaneously also provides the preparation method of this PAM.
Rhizoma Atractylodis polysaccharide is improving the medicine of kuffer cellular immune function or the application in health product.
Described medicine is the medicine of the disease for the treatment of immunodeficiency or tumor.
Described Rhizoma Atractylodis polysaccharide adopts following method to make: adopt the extract that obtains after water extraction with acetone or/and after ethanol repeatedly precipitates, and lyophilization, then carry out separation through DEAE-cellulose column, after water elution, concentrate precipitates with ethanol, is drying to obtain.
The preparation method detailed step of described Rhizoma Atractylodis polysaccharide is as follows: (1) water reflux, extract, three times, merge, and concentrated; (2) for concentrated solution, ethanol, acetone are washed three times repeatedly, and precipitation lyophilization, obtains Rhizoma Atractylodis Macrocephalae crude polysaccharides; (3) Rhizoma Atractylodis Macrocephalae crude polysaccharides is dissolved in a small amount of distilled water, DEAE-cellulose column carries out separation, use respectively distilled water, 0.1mol/L NaCl eluant solution, collect distilled water eluent, (4), by eluent dialysis, dialysis solution is concentrated, add ethanol precipitation, placement is spent the night, and filters, and is drying to obtain Rhizoma Atractylodis polysaccharide (PAM).
In described step (4), also comprise the multiple settling step of ethanol, filtrate is used water dissolution after adding washing with alcohol, filters, and filtrate adds ethanol again to be precipitated again, filters vacuum drying.
Described Rhizoma Atractylodis polysaccharide, before water extraction, adopts 95% alcohol reflux defat.
Rhizoma Atractylodis polysaccharide of the present invention (PAM) is for adopting said method self-control to obtain.Experiment shows that the PAM of suitable concentration can improve normal Kupffer cytophagy, increases the release of NO and TNF-α, and prompting PAM has the effect that activates normal Kupffer cellular immune function.Known, Chinese medicine strengthens immunity and anti-tumor activity effect, has substantial connection with its production of TNF induced-α; NO has panimmunity activity, can antiviral and intracellular pathogen infect, and can kill and wound and suppress tumor growth.Visible, the PAM of optimal dose will be conducive to improve body resistance against diseases undoubtedly, aspect the treatment of the diseases such as immunodeficiency and tumor, have actual application value.
Accompanying drawing explanation
Fig. 1 dextran molecule amount standard reference material gel chromatography figure,
Wherein molecular weight is followed successively by D0:180, D1:2500:D2:4600, D3:7100, D4:10000, D5:21400 from bottom to top;
Fig. 2 PAM molecular weight determination gel chromatography figure,
1.PAM molecular weight is 4300;
Fig. 3 PAM hydrolyzate liquid chromatogram,
1. arabinose; 2. mannose; 3. glucose; 4. galactose.
The specific embodiment
Below in conjunction with experimental example, the present invention is described in further detail.
Embodiment 1 self-control Rhizoma Atractylodis polysaccharide (PAM)
DEAE-cellulose wadding is purchased from Beijing Rui Dahenghui development in science and technology company limited.
Getting Rhizoma Atractylodis Macrocephalae 1000g left and right, be ground into coarse granule, soak 30min, is 95% alcohol heating reflux defat 2h by volume fraction, and filtering residue extracts three times repeatedly with 3 times of water gagings, merges water extraction liquid, concentrated.For concentrated solution, ethanol, acetone are washed three times repeatedly, and precipitation lyophilization, obtains Rhizoma Atractylodis Macrocephalae crude polysaccharides.Getting Rhizoma Atractylodis Macrocephalae crude polysaccharides is dissolved in a small amount of distilled water, DEAE-cellulose column carries out separation, use respectively distilled water, 0.1mol/L NaCl eluant solution, eluent is measured absorbance at 630nm wavelength place by By Anthrone Sulphuric acid method, collect the distilled water part eluent containing polysaccharide.By eluent dialysis, after dialysis solution is concentrated, add ethanol to concentration 75%(V/V), placement is spent the night, filter, precipitation, with after appropriate 75% washing with alcohol, with water dissolution, is filtered, after filtrate adds ethanol to precipitate again, decompress filter, vacuum drying obtains Rhizoma Atractylodis polysaccharide (PAM).
Adopt gel chromatography to measure the molecular weight of polysaccharide, with standard dextran solution, make calibration trace equation, the polysaccharide molecular weight (see figure 1) of utilizing standard curve to ask.Result demonstration, it is homogeneous polysaccharide that Rhizoma Atractylodis polysaccharide obtains PAM after the separation and purification of DEAE-cellulose column, its molecular weight is that 4300Da(is shown in Fig. 2).
For molecular weight determination dextran molecule amount standard reference material (Nat'l Pharmaceutical & Biological Products Control Institute provides, and D0, D1, D2, D3, D4, D5 molecular weight are followed successively by 180,2500,4600,7100,10000,21400).
Get PAM50mg in hydrolysis pipe, add the sulphuric acid 50mL of 5mmol/L, vacuum sealing tube, is placed in 110 ℃ of reaction 6h of baking oven.After having reacted, be cooled to room temperature, add Ba (OH)
2in powder, be 7 with pH value, filter and collect filtrate.Get arabinose, galactose, glucose, mannose reference substance appropriate, be configured to reference substance solution.Adopt high performance liquid chromatography, nh 2 column, differential refraction detector is analyzed, and result shows that the main component of PAM is arabinose, mannose, glucose, galactose (see figure 3).
1. instrument and material
The healthy BALB/C mice of laboratory animal body weight 20～30g, male and female dual-purpose, is provided by the large medical college zoopery of Xi'an traffic center.In constant temperature, periodicity of illumination 12h:12h environment, after raising 1wk, for experiment, 24h fasting before experiment, drinks water arbitrarily.
Medicine and processing Rhizoma Atractylodis polysaccharide (PAM, self-control, purity is greater than 99.9%), from the dry root of Rhizoma Atractylodis Macrocephalae (differentiating to be Atractylodes Macrocephala Koidz. by professor Guo Zengjun of crude drug teaching and research room of Xi'an Jiaotong University Medical College), extract, soluble in water, main component is arabinose, mannose, glucose, galactose.With front, with distilled water, dissolve, through autoclaving, get supernatant after centrifugal.
Reagent actinomycin D, IV Collagenase Type, tetrazolium bromide (MTT) and lipopolysaccharide (LPS) are Sigma product; RPMI-1640 culture medium and new-born calf serum are Gibco product; Tumor necrosis factor α (TNF-α), ALT (ALT), serum glutathione-S-transferase (GST), nitric oxide (NO), lactic acid dehydrogenase (LDH) and acid phosphatase (ACP) test kit are that Bioengineering Research Institute's product is built up in Nanjing.
Instrument Japan Shimadzu UV-2550 ultraviolet-uisible spectrophotometer; DG3022A type enzyme-linked immunosorbent assay instrument.
2. method
2.1 external Kupffer cell induction
Select normal BALB/C mice, pentobarbital sodium subcutaneous injection anesthesia BALB/C Mus, asepticly gets liver, and D-Hank ' s buffer rinses and dehematizes, and removes gallbladder and non-hepatic tissue, puts in the vial of sterilization and shreds with eye scissors, and hepatic tissue blocking is cut into 1mm
3size.Every gram of hepatic tissue adds 10mL0.05% pronase e solution, gently piping and druming digestion 10min.200 order stainless steel meshs filter, the centrifugal 10min of 100 * g, abandon supernatant, D-Hank ' s buffer solution for cleaning 2 times, 30%～70%Percoll separating medium gradient centrifugation 15min(1000 * g), get intermediate layer, RPMI-1640 liquid cleans 2 times, and 10%FCS is incubated at glass culture bottle, after 4h, with RPMI-1640, rinse gently 3 times and remove non-adherent cell, attached cell is the Kupffer cell of separation and purification.Adopt the survival rate of Kupffer cell in the blue Determination Staining Mouse Liver of platform dish to be greater than 95%, result shows, its cell survival rate meets the demands.Peroxidase stain discriminating, Kupffer cell purity is greater than 90%.Cultivate 20h, after Kupffer cell attachment, remove supernatant, with containing 10% new-born calf serum RPMI-1640 culture medium (10%NBS-1640 liquid), PAM doubling dilution is become to series mass concentration (800,400,200,100,50 and 25mgL
-1) act on Kupffer cell.The negative contrast of 10%NBS-1640 liquid, take whole mass concentration as 40mgL
-1the positive contrast of lipopolysaccharide (LPS), establish 4 multiple holes for every group, continue to cultivate after 24h, measure phagocytic function; Collect respectively supernatant ,-80 ℃ of preservations, NO to be measured, TNF-α and LDH.
2.2 the effect of external PAM to hepatic parenchymal cells
Select normal BALB/C mice, with chloral hydrate (3.5%, 1mL/100g, ip) fixation postures, sterilization skin of chest abdomen after anesthesia, open peritoneum, after free portal vein and postcava, insert No. 22 capable portal catheterizations of conduit apart from the about 1cm of hepatic portal place, it is standby that another conduit is fixed in postcava insertion.37 ℃ of pre-temperature D-Hanks ' liquid are with the perfusion of 25mL/min flow velocity, and after several minutes, visible liver expands, and color even is pale.Cut off postcava and emit hematocele hydrops, portal vein is used instead containing the Hanks liquid of 0.025%IV Collagenase Type and is filled with rapidly about 10min.Treat that liver toughness disappears, compressing is sunk and is cut off liver week ligament while being difficult for recovering, and cuts liver, is placed in the PBS of 4 ℃ of pre-coolings; Scratch liver tunicle, use glass minute hand combing liver gently, to impel cell to disperse.Collecting cell suspension is placed on 400 order nylon wires and filters, filtrate is liver cell suspension. with the PBS of 4 ℃, wash liver cells 3 times, cell suspension is laid on 60%Percoll liquid level, 4 ℃, the centrifugal 15min of 200g, collecting precipitation, obtains the hepatocyte of separation and purification, adds the RPMI-1640 culture fluid containing 10% hyclone (10%FCS).Put 37 ℃, 5%CO
2in incubator, cultivate, within every 24 hours, change culture medium.After adopting 0.25% trypsinization 3～5min, carry out passage.Adopting the survival rate of the blue Determination Staining mouse liver cell of platform dish is 96 ± 3%, and result shows, its cell survival rate meets the requirement of In vitro culture.With " collagen gel sandwich ", cultivate Mouse Liver parenchyma, add PAM, whole mass concentration 800,400,200,100,50 and 25mgL
-1, every concentration is established 4 multiple holes, and 10%NBS-1640 liquid is blank.Cultivate after 24h, collect supernatant, LDH to be measured.
Kupffer cell induction in 2.3 bodies
2.4 phagocytic function
By Kupffer cell suspension with 1.25 * 10
5individual mL
-1add 96 well culture plates, obtain cell monolayer, abandon supernatant, add 0.072% dimethyl diaminophenazine chloride 0.1mL/ hole, cultivate 30min, abandon dimethyl diaminophenazine chloride, and wash away the dimethyl diaminophenazine chloride of not engulfed with PBS, finally add cell pyrolysis liquid 0.1molL
-1glacial acetic acid-dehydrated alcohol (1: 1), hold over night is surveyed A in microplate reader
540the absorption value at place.
2.5TNF-α, NO, LDH, ACP, the mensuration of sALT and sGST
Reference reagent box is measured.
2.6 statistical procedures
Data all represent with Mean ± SD, and between two groups, mean relatively adopts t check.
3. result
3.1PAM to Kupffer cell and hepatic parenchymal cells culture supernatant LDH content influence
The damage of LDH leakage reflection medicine to cultured cell.As shown in table 1, the external PAM that gives, each dosage group LDH leakage, without obvious rising, shows that PAM treated in vitro does not have direct detrimental effect to Kupffer cell and hepatic parenchymal cells.
Table 1PAM is to Kupffer cell and hepatic parenchymal cells culture supernatant LDH content influence (n=4, Mean ± SD)
3.2 impacts of external PAM on Kupffer cellular immune function
As shown in table 2, PAM is at 31.25～250mgL
-1, it is active that concentration dependent strengthens Kupffer cytophagy, and with negative control group comparison, increasing degree is 10.5%～73.7% (P<0.05, P<0.01); At 500～2000mgL
-1activate the phagocytic capacity no longer presents concentration dependent.
Table 2PAM is on the impact of the Kupffer cellular immune function of In vitro culture (n=5, Mean ± SD)
Note: * and 10%NBS-1640 group be P<0.05 relatively, * * and 10%NBS-1640 group be P<0.01 relatively
PAM (25～400mgL
-1) can make Kupffer cell be concentration dependent rising (P<0.05, P<0.01), 400mgL to the secretion of NO
-1the amount that induction produces NO is (0.71 ± 0.02) UL
-1, PAM concentration surpasses 400mgL
-1it is substantially constant that induction produces the amount of NO.
PAM (25～400mgL
-1) can obviously promote the external generation (P<0.05, P<0.01) of TNF-α.400mgL
-1pAM makes TNF-alpha levels increase by 119%, PAM concentration compared with matched group to be increased, and TNF-α produces and further do not increase.
3.3 the impact of PAM on mice Kupffer cellular immune function in body
As shown in table 3, in body, give PAM and can strengthen the red phagocytic function of Kupffer cell centering.PAM0.1,0.5 and 1.0gkg
-1compared significant difference (P<0.05, P<0.01) with matched group; And at 0.05～1.0gkg
-1, its phagocytic activity is dose dependent increases (r=0.818).
PAM increases ACP activity in Kupffer cell, at 0.05～1.0gkg
-1active increasing with dosage increase has dependency (r=0.763); 0.5gkg
-1time, ACP activity is 1.4 times of matched groups, PAM concentration is greater than 0.5gkg
-1time ACP activity no longer increase.
PAM can make Kupffer cell NO produce to be dose dependent to raise (r=0.834), 0.1,0.5 and 1.0gkg
-1dosage group has significant (P<0.05, P<0.01).
PAM vivo medicine-feeding 0.1,0.5 and 1.0gkg
-1, can make the generation of TNF-α increase (P<0.05, P<0.01), wherein give PAM1.0gkg
-1can make TNF-α increase by 215%.
In table 3 body, PAM is on the impact of mice Kupffer cellular immune function (n=5, Mea ± SD)
Note: * and matched group be P<005 relatively, * * and matched group be P<001 relatively.
The impact of 3.4PAM on sALT and sGST
PAM is at 0.5～1.0gkg
-1to sALT and sGST activity influence and matched group there was no significant difference (table 4).
Table 4PAM is on the impact of sALT and sGST (n=5, Mean ± SD)
4. discuss
Kupffer cell is macrophage in liver, can engulf large particulate matter (as antibacterial, tumor cell), also can gulp down drink solable matter (as enterotoxin, bacterial antigens), and discharge the panimmunity factors such as NO and TNF-α.This style is interior, in vitro results all shows, the PAM of suitable concentration can improve normal Kupffer cytophagy, increases the release of NO and TNF-α, and prompting PAM has the effect that activates normal Kupffer cellular immune function.Known, Chinese medicine strengthens immunity and anti-tumor activity effect, has substantial connection with its production of TNF induced-α
[6]; NO has panimmunity activity, can antiviral and intracellular pathogen infect, and can kill and wound and suppress tumor growth.Visible, the PAM of optimal dose will be conducive to improve body resistance against diseases undoubtedly, aspect the treatment of the diseases such as immunodeficiency and tumor, have actual application value.
Claims (6)
1. Rhizoma Atractylodis polysaccharide is improving the medicine of kuffer cellular immune function or the application in health product.
2. application according to claim 1, described medicine is the medicine of the disease for the treatment of immunodeficiency or tumor.
3. application according to claim 1, described Rhizoma Atractylodis polysaccharide adopts following method to make: adopt the extract that obtains after water extraction with acetone or/and after ethanol repeatedly precipitates, lyophilization, through DEAE-cellulose column, carry out separation again, after water elution, concentrate precipitates with ethanol, is drying to obtain.
4. application according to claim 3, the preparation method detailed step of described Rhizoma Atractylodis polysaccharide is as follows: (1) water reflux, extract, three times, merge, concentrated; (2) for concentrated solution, ethanol, acetone are washed three times repeatedly, and precipitation lyophilization, obtains Rhizoma Atractylodis Macrocephalae crude polysaccharides; (3) Rhizoma Atractylodis Macrocephalae crude polysaccharides is dissolved in a small amount of distilled water, DEAE-cellulose column carries out separation, use respectively distilled water, 0.1mol/LNaCl eluant solution, collect distilled water eluent, (4), by eluent dialysis, dialysis solution is concentrated, add ethanol precipitation, placement is spent the night, and filters, and is drying to obtain Rhizoma Atractylodis polysaccharide (PAM).
5. application according to claim 4, also comprises the multiple settling step of ethanol in described step (4), filtrate is used water dissolution after adding washing with alcohol, filters, and filtrate adds ethanol again to be precipitated again, filters vacuum drying.
6. application according to claim 3, described Rhizoma Atractylodis polysaccharide, before water extraction, adopts 95% alcohol reflux defat.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113185618A (en) * | 2021-04-28 | 2021-07-30 | 南京中医药大学 | Anti-alcoholic liver injury rhizoma atractylodis macrocephalae polysaccharide and preparation method and application thereof |
| CN114539439A (en) * | 2022-04-01 | 2022-05-27 | 哈尔滨工业大学 | Atractylodes macrocephala polysaccharide AMP1-1, and extraction method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1397566A (en) * | 2002-04-05 | 2003-02-19 | 中国科学院上海有机化学研究所 | Atractylodes polyose with antineoplastic and immunoregualting functions and its preparing process and application |
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| CN1397566A (en) * | 2002-04-05 | 2003-02-19 | 中国科学院上海有机化学研究所 | Atractylodes polyose with antineoplastic and immunoregualting functions and its preparing process and application |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113185618A (en) * | 2021-04-28 | 2021-07-30 | 南京中医药大学 | Anti-alcoholic liver injury rhizoma atractylodis macrocephalae polysaccharide and preparation method and application thereof |
| CN113185618B (en) * | 2021-04-28 | 2022-06-07 | 南京中医药大学 | Anti-alcoholic liver injury rhizoma atractylodis macrocephalae polysaccharide and preparation method and application thereof |
| CN114539439A (en) * | 2022-04-01 | 2022-05-27 | 哈尔滨工业大学 | Atractylodes macrocephala polysaccharide AMP1-1, and extraction method and application thereof |
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