CN104829737A - Crude raspberry leaf polysaccharide, and preparation method and application thereof - Google Patents
Crude raspberry leaf polysaccharide, and preparation method and application thereof Download PDFInfo
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Abstract
The invention specifically relates to a crude raspberry leaf polysaccharide, and a preparation method and application thereof, belonging to the field of traditional Chinese medicine. The crude raspberry leaf polysaccharide is extracted from leaves of raspberry through steps consisting of hot water reflux extraction, decoloring, alcohol precipitation and the like; and the crude raspberry leaf polysaccharide can substantially inhibit release of LPS-induced RAW264.7 macrophage NO in vitro and substantially suppress expression of LPS-induced RAW264.7 macrophages TNF-alpha, iNOS and IL-6mRNA. Moreover, the crude raspberry leaf polysaccharide is further purified by using ion exchange column chromatography, and three components are obtained, wherein the component with highest content is refined raspberry leaf polysaccharide, the refined raspberry leaf polysaccharide is a heteropolysaccharide containing five monosaccharides, and a sugar residue mol ratio of rhamnose to arabinose to xylose to glucose to galactose is 2.47: 4.75: 4.12: 1: 2.48.
Description
Technical field
The invention belongs to the field of Chinese medicines, be specifically related to a kind of leaf of Palmleaf Raspberry Crude polysaccharides and preparation method thereof and application.
Background technology
One of Antiinflammatory Regimen of the most promising current alternative classical antibiotic therapy uses the immunomodulator that can strengthen host defense.Existing known panimmunity conditioning agent, comprises mammalian proteins, as: gamma-interferon, granulocyte colony-stimulating factor and granulocyte-macrophage colony stimutaing factor and the material from microorganism separation and purification.For the compound of immune bacterial polysaccharides and synthetic, its untoward reaction and side effect have caused people extensively to note.The polysaccharide in most of higher plant source is the material had no adverse reaction, and can not produce larger side effect to body, therefore, the polysaccharide be separated from plant causes great concern in biomedicine.To the mechanism of action of these active substances also at development, wherein the immunologic mechanism of polysaccharide to non-specific induction is more subject to people's attention.Research finds, vegetable polysaccharides is antitumor, the immuno-stimulating mechanism of sterilization and other treatment effect has been come by the adjustment of complement system and the stimulation of scavenger cell.Research finds, the polysaccharide of plant origin embodies various useful pharmacological action by regulating the immunologic function of scavenger cell.Vegetable polysaccharides is anti-inflammatory, antitumor and promote the optimal vaccine candidate medicine of wound healing.
Raspberry (Rubus chingii Hu), i.e. Rubus chingii is the Rosaceae (Rosaceae) rubus, main product in Zhejiang, Fujian, this external Anhui, Jiangxi, Jiangsu etc. are economized also has distribution, because it is mainly distributed in East China also known as rubus chingii Hu.Raspberry is that Pharmacopoeia of the People's Republic of China version one in 2010 is included, and with its prematurity dry fruit for medicinal part, has effect of kidney tonifying, controlling nocturnal emission with astringent drugs, contracting urine, for enuresis due to deficiency of the kidney, frequent micturition, positive paralysis premature ejaculation, emission and spermatorrhea etc.Research for raspberry fruit polysaccharide is more, but does not but have for the research of raspberry leaf polysaccharide.
Summary of the invention
In order to overcome the deficiencies in the prior art and shortcoming, primary and foremost purpose of the present invention is the preparation method providing a kind of leaf of Palmleaf Raspberry Crude polysaccharides.
Another object of the present invention is to the leaf of Palmleaf Raspberry Crude polysaccharides providing above-mentioned preparation method to prepare.
Another object of the present invention is the application providing above-mentioned leaf of Palmleaf Raspberry Crude polysaccharides.
Object of the present invention is realized by following proposal:
A preparation method for leaf of Palmleaf Raspberry Crude polysaccharides, comprises following steps:
(1) sieve after raspberry (Rubus chingii Hu) crushing up leaves, obtain leaf of Palmleaf Raspberry powder; 75 DEG C ~ 95 DEG C hot water returns extract Rubus chingii leaf powder, then that extracting solution is centrifugal, get supernatant liquid, filter 2 ~ 4 times, by filtrate reduced in volume, obtain polysaccharide soln;
(2) mix by polysaccharide soln with through pretreated macroporous adsorbent resin, decolour in 40 DEG C ~ 60 DEG C water-bath 2 ~ 4h, then filter out polysaccharide soln, and the polysaccharide adsorbed in resin is repeatedly washed out with water, merged by polysaccharide soln after decolouring, concentrating under reduced pressure, obtains concentrated liquid glucose;
(3) ethanol adding 3 ~ 5 times of volumes in concentrated liquid glucose carries out alcohol precipitation, then centrifugal, abandoning supernatant, and collecting precipitation thing is also dry, obtains leaf of Palmleaf Raspberry Crude polysaccharides (L-Ps);
The ratio of water to material that hot water return described in step (1) extracts is (10:1) ~ (30:1);
The time that hot water return described in step (1) extracts is 2 ~ 4h, and the number of times that hot water return extracts is 2 ~ 4 times;
Centrifugal rotating speed described in step (1) is 3000 ~ 5000rpm/min; The described centrifugal time is 8 ~ 12min;
Macroporous adsorbent resin described in step (2) is preferably D354FD resin;
The pretreated method of the macroporous adsorbent resin described in step (2) is: be first soaked in water macroporous adsorbent resin 12 ~ 24h, then successively with massfraction be 5% hydrochloric acid soln and massfraction be 5% sodium hydroxide solution soak 0.5 ~ 3h separately, filter, be washed to neutrality, obtain through pretreated macroporous adsorbent resin;
The temperature of step (1) and the concentrating under reduced pressure described in (2) is 40 ~ 60 DEG C;
The volume fraction of the ethanol described in step (3) is 70% ~ 100%;
The temperature of the alcohol precipitation described in step (3) is 0 ~ 4 DEG C, and the time of alcohol precipitation is 8 ~ 24h;
Centrifugal rotating speed described in step (3) is 3000 ~ 5000rpm/min; The described centrifugal time is 12 ~ 15min;
The temperature of the drying described in step (3) is 40 ~ 60 DEG C;
A kind of leaf of Palmleaf Raspberry Crude polysaccharides (L-Ps), is prepared by above-mentioned preparation method;
A kind of raspberry folic acid polysaccharide (L-Ps-1), obtains by being further purified by above-mentioned leaf of Palmleaf Raspberry Crude polysaccharides;
Described raspberry folic acid polysaccharide (L-Ps-1) is homogeneous components, and its molecular weight (Mn) is 12686Da;
Described raspberry folic acid polysaccharide (L-Ps-1) is for comprising the mixed polysaccharide of five kinds of monose, and wherein, saccharide residue mol ratio is: rhamnosyl: pectinose: wood sugar: glucose: semi-lactosi=2.47:4.75:4.12:1:2.48;
Described raspberry folic acid polysaccharide (L-Ps-1) prepares as follows:
(1) will proceed to after 40 ~ 60 DEG C of rotary evaporation bubble removings in Z-type chromatography column through pretreated DEAE-52 resin, even to make filler fill post with constant flow pump pumping distilled water, after balance, flow velocity 5 ~ 10s/ drips;
(2) leaf of Palmleaf Raspberry Crude polysaccharides is mixed with polysaccharide soln, proceed in chromatography column, use distilled water, 0.05mol/L NaCl, 0.1mol/L NaCl, 0.3mol/L NaCl, 0.5mol/L NaCl solution wash-out successively, flow velocity 5 ~ 10s/ drips, collect each stream part respectively, phend-sulphuric acid tracing detection elutriant, measure the absorbancy under 490nm, draw elution curve, the elutriant in same absorption peak merges; Then concentrated by rotary evaporation, vacuum lyophilization, obtains three components after DEAE-52 ion-exchange chromatography, and wherein, the component that content is the highest is raspberry folic acid polysaccharide (L-Ps-1);
The pretreated method of DEAE-52 resin described in step (1) is: 4 ~ 30 DEG C, DEAE-52 resin water is soaked 12 ~ 24h; 0.5 ~ 2h is soaked again with the hydrochloric acid soln of 0.5mol/L; The NaOH solution of 0.5mol/L soaks 0.5 ~ 2h, filters, is washed to neutrality, obtains through pretreated DEAE-52 resin;
The temperature of the concentrated by rotary evaporation described in step (2) is 40 ~ 60 DEG C;
Described leaf of Palmleaf Raspberry Crude polysaccharides or raspberry folic acid polysaccharide are preparing the application in anti-inflammatory drug;
Described leaf of Palmleaf Raspberry Crude polysaccharides or raspberry folic acid polysaccharide can be used as novel anti-inflammatory drug, are applied to the treatment of inflammation.
A kind of anti-inflammatory drug, containing above-mentioned leaf of Palmleaf Raspberry Crude polysaccharides or raspberry folic acid polysaccharide;
Principle of the present invention: the polysaccharide of plant origin embodies various useful pharmacological action by regulating the immunologic function of scavenger cell.The present invention finds and confirmation vegetable polysaccharides L-Ps has significant anti-inflammatory activity, and the using dosage of vegetable polysaccharides L-Ps is low, and the toxic side effect under same dosage is also lower.
The present invention has following advantage and effect relative to prior art:
(1) release of the Late Cambrian leaf of Palmleaf Raspberry Crude polysaccharides of the present invention RAW264.7 scavenger cell NO that can significantly suppress LPS to induce in vitro.
(2) expression of Late Cambrian leaf of Palmleaf Raspberry Crude polysaccharides of the present invention RAW264.7 scavenger cell TNF-α, iNOS and IL-6mRNA that can significantly suppress LPS to induce in vitro.
(3) dosage of leaf of Palmleaf Raspberry Crude polysaccharides is lower, and endotoxic lower in effective pharmaceutical quantities scope.
Accompanying drawing explanation
Fig. 1 is the ultraviolet scanning spectrum figure of the leaf of Palmleaf Raspberry Crude polysaccharides that embodiment 1 prepares.
Fig. 2 is the infrared spectrogram of the leaf of Palmleaf Raspberry Crude polysaccharides that embodiment 1 prepares.
Fig. 3 is the ion-exchange chromatography elution curve of embodiment 5.
Fig. 4 is the GPC figure of the raspberry folic acid polysaccharide that embodiment 5 prepares.
Fig. 5 is the effect diagram of leaf of Palmleaf Raspberry Crude polysaccharides to the RAW264.7 scavenger cell release NO that LPS induces.
Fig. 6 is the effect diagram of leaf of Palmleaf Raspberry Crude polysaccharides to the RAW264.7 scavenger cell TNF-α mrna expression that LPS induces, wherein, and ##: compare with control group, P<0.01.
Fig. 7 is the effect diagram of leaf of Palmleaf Raspberry Crude polysaccharides to the RAW264.7 scavenger cell iNOS mrna expression that LPS induces, wherein, and ##: compare with control group, P<0.01.
Fig. 8 is the effect diagram that leaf of Palmleaf Raspberry Crude polysaccharides is expressed the RAW264.7 scavenger cell IL-6mRNA that LPS induces, wherein, and ##: compare with control group, P<0.01.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
In embodiment, mouse macrophage RAW 264.7 is purchased from life science institute cellular resources center, Chinese Academy of Sciences Shanghai; Rubus chingii leaf picks up from Zhejiang farmers'.
Embodiment 1 prepares leaf of Palmleaf Raspberry Crude polysaccharides (L-Ps) from Rubus chingii leaf
(1) cross 20 mesh sieves after Rubus chingii crushing up leaves, take 100g, use distilled water heating and refluxing extraction, wherein, ratio of water to material is 20:1, and refluxing extraction temperature is 75 DEG C, and reflux extracting time is 4h, refluxing extraction number of times 3 times; Then united extraction liquid, under 4000rpm/min, centrifugal 10min, gets supernatant liquid, and filter paper filtering 3 times, is evaporated to 200mL with Rotary Evaporators by filtrate, obtains polysaccharide soln at 40 DEG C;
(2) D354FD resin is first used distilled water immersion 12h, then 0.5h is soaked separately with the sodium hydroxide solution that hydrochloric acid soln, massfraction that massfraction is 5% are 5% successively, 200 order filtered through gauze, distilled water is washed till neutrality, obtains through pretreated D354FD resin;
(3) polysaccharide soln step (1) prepared and 200mL are through pretreated D354FD mixed with resin, be placed in 50 DEG C of thermostat water bath water bath with thermostatic control 3h to decolour, interval is stirred, then polysaccharide soln is gone out with 200 object filter-cloth filterings, and the polysaccharide adsorbed in resin is repeatedly washed out with water, polysaccharide liquid after decolouring is merged, at 40 DEG C, is evaporated to 200mL with Rotary Evaporators, obtains concentrated liquid glucose;
(4) volume fraction adding 4 times of volumes in the concentrated liquid glucose prepared in step (3) is the ethanol of 95%, limit edged magnetic agitation, alcohol precipitation 8h under 4 DEG C of conditions, then centrifugation 15min under 4000rpm/min, abandoning supernatant, take out throw out in 45 DEG C of oven dry, obtain leaf of Palmleaf Raspberry Crude polysaccharides (L-Ps).
Embodiment 2 prepares leaf of Palmleaf Raspberry Crude polysaccharides (L-Ps) from Rubus chingii leaf
(1) cross 20 mesh sieves after Rubus chingii crushing up leaves, take 100g, then distilled water heating and refluxing extraction, wherein, ratio of water to material 20:1, refluxing extraction temperature is 85 DEG C, and reflux extracting time is 3h, refluxing extraction number of times 2 times; Then united extraction liquid, under 3000rpm/min, centrifugal 8min, gets supernatant liquid, and filter paper filtering 2 times, is evaporated to 200mL with Rotary Evaporators by filtrate, obtains polysaccharide soln at 50 DEG C;
(2) D354FD resin is first used distilled water immersion 24h, then 2h is soaked separately with the sodium hydroxide solution that hydrochloric acid soln, massfraction that massfraction is 5% are 5% successively, 200 order filtered through gauze, distilled water is washed till neutrality, obtains through pretreated D354FD resin;
(3) polysaccharide soln step (1) prepared and 200mL are through pretreated D354FD mixed with resin, be placed in 40 DEG C of thermostat water bath water bath with thermostatic control 2h to decolour, interval is stirred, then polysaccharide soln is gone out with 200 object filter-cloth filterings, and the polysaccharide adsorbed in resin is repeatedly washed out with water, polysaccharide liquid after decolouring is merged, at 50 DEG C, is evaporated to 200mL with Rotary Evaporators, obtains concentrated liquid glucose;
(4) volume fraction adding 3 times of volumes in the concentrated liquid glucose prepared in step (3) is the ethanol of 95%, limit edged magnetic agitation, alcohol precipitation 12h under 0 DEG C of condition, then centrifugation 12min under 3000rpm/min, abandoning supernatant, take out throw out in 50 DEG C of oven dry, obtain leaf of Palmleaf Raspberry Crude polysaccharides (L-Ps).
Embodiment 3 prepares leaf of Palmleaf Raspberry Crude polysaccharides (L-Ps) from Rubus chingii leaf
(1) cross 20 mesh sieves after Rubus chingii crushing up leaves, take 100g, use distilled water heating and refluxing extraction, wherein, ratio of water to material is 20:1, and refluxing extraction temperature is 95 DEG C, reflux extracting time is 2h, refluxing extraction number of times 4 times, then united extraction liquid, centrifugal 12min under 5000rmp/min, get supernatant liquid, filter paper filtering 4 times, is evaporated to 200mL with Rotary Evaporators by filtrate, obtains polysaccharide soln at 60 DEG C;
(2) D354FD resin is first used distilled water immersion 18h, then 3h is soaked separately with the sodium hydroxide solution that hydrochloric acid soln, massfraction that massfraction is 5% are 5% successively, 200 order filtered through gauze, distilled water is washed till neutrality, obtains through pretreated D354FD resin;
(3) by the polysaccharide soln for preparing in (1) and 200mL through pretreated D354FD mixed with resin, be placed in 60 DEG C of thermostat water bath water bath with thermostatic control 4h to decolour, interval is stirred, polysaccharide soln is gone out afterwards with 200 object filter-cloth filterings, and the polysaccharide adsorbed in resin is repeatedly washed out with water, polysaccharide liquid after decolouring is merged, at 60 DEG C, is evaporated to 200mL with Rotary Evaporators, obtains concentrated liquid glucose;
(4) volume fraction adding 5 times of volumes in the concentrated liquid glucose prepared in step (3) is the ethanol of 95%, limit edged magnetic agitation, alcohol precipitation 24h under 4 DEG C of conditions, then centrifugation 14min under 5000rpm/min, abandoning supernatant, take out throw out in 60 DEG C of oven dry, obtain leaf of Palmleaf Raspberry Crude polysaccharides (L-Ps).
Embodiment 4 UV scanning collection of illustrative plates and Infrared spectrum scanning
(1) leaf of Palmleaf Raspberry Crude polysaccharides (L-Ps) 4mg prepared in Example 1 ~ 3 respectively, is mixed with the polysaccharide soln of 1mg/mL, take distilled water as contrast, in its absorbance of 190 ~ 500nm place survey.
(2) leaf of Palmleaf Raspberry Crude polysaccharides (L-Ps) 2mg prepared in Example 1 ~ 3 respectively, with KBr mixing porphyrize uniformly lamelliform, at 4000 ~ 400cm
-1infrared scan is carried out in wave-number range.
Interpretation of result:
(1) polysaccharide ultraviolet spectral analysis result: the leaf of Palmleaf Raspberry Crude polysaccharides prepared in embodiment 1 all has stronger absorption at 200 ~ 280nm, show may contain unsaturated carbonyl and carboxyl in sample, there is stronger absorption peak at 266nm place, show the nucleic acid containing conjugation in sample or protein (Fig. 1).The detected result of embodiment 2 and embodiment 3 is with embodiment 1.
(2) polysaccharide results of IR: the leaf of Palmleaf Raspberry Crude polysaccharides prepared in embodiment 1 is all at wave number 3345cm
-1the strong absorption peak at place is between carbohydrate molecule or intramolecular O-H stretching vibration peak, at wave number 2933cm
-1the absorption peak that place exists is the C-H vibration absorption peak of methyne or methyl, at wave number 1430 ~ 1200cm
-1the absorption peak at place is the C-O stretching vibration peak of carboxyl, and the characteristic peak of these three groups of carbohydrates can judge that the leaf of Palmleaf Raspberry Crude polysaccharides prepared in embodiment 1 is polysaccharide.At wave number 1000 ~ 1200cm
-1the absorption peak at place is the vibration peak of uronic acid, at wave number 1617cm
-1the relatively weak absorption peak at place is the characteristic IR absorbance peaks (Fig. 2) of protein.These results further illustrate the leaf of Palmleaf Raspberry Crude polysaccharides prepared in embodiment 1 and contain polysaccharide, protein and sugar aldehydic acid.The detected result of embodiment 2 and embodiment 3 is with embodiment 1.
The preparation of embodiment 5 raspberry folic acid polysaccharide (L-Ps-1)
(1) DEAE-52 pre-treatment: the DEAE-52 getting 70g, 24h is soaked with distilled water 4 DEG C, then careful upper water to be poured out, removing impurity, then with the hydrochloric acid soln immersion 0.5h of 0.5mol/L, upper strata acid solution is carefully poured out, suction filtration is eluted to neutrality with distilled water after dry, then soaks 0.5h by the NaOH solution of 0.5mol/L, carefully pours out upper strata alkali lye, suction filtration is washed till neutrality with distilled water after dry, for subsequent use;
(2) will proceed in the Z-type chromatography column of 2.6 × 30cm specification through pretreated DEAE-52 glass stick drainage after 48 DEG C of rotary evaporation bubble removings, even to make filler fill post with constant flow pump pumping distilled water, after balance, 7s/ drips;
(3) the leaf of Palmleaf Raspberry Crude polysaccharides (L-Ps) prepared in 100mg embodiment 1 is taken, be mixed with polysaccharide soln, glass stick is drained in chromatography column, use distilled water, 0.05mol/L NaCl, 0.1mol/LNaCl, 0.3mol/L NaCl, 0.5mol/L NaCl solution wash-out successively, automatic collection each stream part, about 5mL collected by every root pipe, phend-sulphuric acid tracing detection elutriant, measure the absorbancy at 490nm place, draw elution curve, the elutriant in same absorption peak merges; Then elutriant 40 DEG C of concentrated by rotary evaporations, vacuum lyophilization, wherein, L-Ps obtains three components after DEAE-52 ion-exchange chromatography, the component (48.0%) that content is the highest is raspberry folic acid polysaccharide (L-Ps-1), wherein, Fig. 3 is the ion-exchange chromatography elution curve of the present embodiment.
The preparation of embodiment 6 raspberry folic acid polysaccharide (L-Ps-1)
(1) DEAE-52 pre-treatment: the DEAE-52 getting 70g, 12h is soaked with distilled water 20 DEG C, then careful upper water to be poured out, removing impurity, then with the hydrochloric acid soln immersion 1h of 0.5mol/L, upper strata acid solution is carefully poured out, suction filtration is eluted to neutrality with distilled water after dry, then soaks 1h by the NaOH solution of 0.5mol/L, carefully pours out upper strata alkali lye, suction filtration is washed till neutrality with distilled water after dry, for subsequent use;
(2) will proceed in the Z-type chromatography column of 2.6 × 30cm specification through pretreated DEAE-52 glass stick drainage after 48 DEG C of rotary evaporation bubble removings, even to make filler fill post with constant flow pump pumping distilled water, after balance, 7s/ drips;
(3) the leaf of Palmleaf Raspberry Crude polysaccharides (L-Ps) prepared in 100mg embodiment 1 is taken, be mixed with polysaccharide soln, glass stick is drained in chromatography column, use distilled water, 0.05mol/L NaCl, 0.1mol/LNaCl, 0.3mol/L NaCl, 0.5mol/L NaCl solution wash-out successively, automatic collection each stream part, about 5mL collected by every root pipe, phend-sulphuric acid tracing detection elutriant, measure the absorbancy at 490nm place, draw elution curve, the elutriant in same absorption peak merges; Then elutriant 50 DEG C of concentrated by rotary evaporations, vacuum lyophilization, wherein, L-Ps obtains three components after DEAE-52 ion-exchange chromatography, the component (48.0%) that content is the highest is raspberry folic acid polysaccharide (L-Ps-1), wherein, the ion-exchange chromatography wash-out result of the present embodiment is with embodiment 5.
The preparation of embodiment 7 raspberry folic acid polysaccharide (L-Ps-1)
(1) DEAE-52 pre-treatment: the DEAE-52 getting 70g, 20h is soaked with distilled water 25 DEG C, then careful upper water to be poured out, removing impurity, then with the hydrochloric acid soln immersion 0.8h of 0.5mol/L, upper strata acid solution is carefully poured out, suction filtration is eluted to neutrality with distilled water after dry, then soaks 0.8h by the NaOH solution of 0.5mol/L, carefully pours out upper strata alkali lye, suction filtration is washed till neutrality with distilled water after dry, for subsequent use;
(2) will proceed in the Z-type chromatography column of 2.6 × 30cm specification through pretreated DEAE-52 glass stick drainage after 48 DEG C of rotary evaporation bubble removings, even to make filler fill post with constant flow pump pumping distilled water, after balance, 7s/ drips;
(3) the leaf of Palmleaf Raspberry Crude polysaccharides (L-Ps) prepared in 100mg embodiment 1 is taken, be mixed with polysaccharide soln, glass stick is drained in chromatography column, use distilled water, 0.05mol/L NaCl, 0.1mol/LNaCl, 0.3mol/L NaCl, 0.5mol/L NaCl solution wash-out successively, automatic collection each stream part, about 5mL collected by every root pipe, phend-sulphuric acid tracing detection elutriant, measure the absorbancy at 490nm place, the elutriant drawn in the same absorption peak of elution curve merges; Then elutriant 60 DEG C of concentrated by rotary evaporations, vacuum lyophilization,
Wherein, L-Ps obtains three components after DEAE-52 ion-exchange chromatography, the component (48.0%) that content is the highest is raspberry folic acid polysaccharide (L-Ps-1), and wherein, the ion-exchange chromatography wash-out result of the present embodiment is with embodiment 5.
The molecular weight of embodiment 8L-Ps-1, Purity and monosaccharide composition analysis
(1) high performance liquid chromatograph is adopted to measure the molecular weight of the raspberry folic acid polysaccharide (L-Ps-1) that embodiment 5 ~ 7 prepares and purity thereof respectively.Chromatographic condition: TSK G-5000PXL column (7.8 × 300mm) and TSK G-3000PXL column (7.8 × 300mm) series connection, moving phase is the KH of 0.02mol/L
2pO
4buffered soln, flow velocity is 0.6mL/min, 2414 Composition distribution, column temperature 35 DEG C.
(2) take raspberry folic acid polysaccharide (L-Ps-1) 10mg that embodiment 5 ~ 7 prepares respectively and be placed in 5mL ampere pipe, add the trifluoroacetic acid solution 4mL of 2mol/L, alcohol blast burner seals, 110 DEG C of hydrolysis 6h, afterwards by hydrolyzation sample 48 DEG C of concentrating under reduced pressure evaporates to dryness, add appropriate volume methyl alcohol, then 48 DEG C of concentrating under reduced pressure evaporates to dryness, so repeat 3 times to sample is neutrality.Get 10mg glucose respectively, Fucose, rhamnosyl, wood sugar, seminose, pectinose, semi-lactosi standard substance and each hydrolysate are in 10mL screw socket pipe, add 10mg oxammonium hydrochloride, 1mL pyridine, 30min is reacted under 90 DEG C of water bath condition, in reaction process every 5min vibration once, acetic anhydride 1mL is added after naturally cooling to room temperature, reaction 30min is continued again under 90 DEG C of water bath condition, in reaction process every 5min vibration once, room temperature is naturally cooled to after taking-up, final generation has volatile sugared nitrile acetic ester derivative, cross film (0.22 μm) process, directly carry out GC analysis afterwards, contrast with the retention time of standard substance per sample, obtain the monose composition of various sample polysaccharide, and calculate various monose weight percent according to the area ratio at each peak.Chromatographic condition: Aglient6890N gas chromatograph; DB-1701 capillary column (30m × 0.25m, 0.25 μm); Temperature of vaporization chamber 250 DEG C, detector temperature 300 DEG C, column temperature 180 DEG C; Temperature programming: 180 (2min) are warmed up to 220 DEG C with the speed in 2 DEG C/min, keeps in 1min, and then is warmed up to 250 DEG C with the speed in 5 DEG C/min, and keep in 2min.Detector: fid detector; Flow rate of carrier gas: 1mL/min; Purge: 4.0mL/min.
Interpretation of result:
(1) the GPC figure of L-Ps-1 that embodiment 5 prepares is single symmetrical peak, show that L-Ps-1 is homogeneous polysaccharide, its the equal molecular mass of number (Mn) is 12686Da, weight average molecular mass (Mw) is 16073Da, and peak position molecular mass (Mp) is 17415Da (Fig. 4).Embodiment 6 and 7 result is with embodiment 5.
(2) according to area normalization method, the ratio of peak area equals contained material mass ratio, according to the amount of substance being each material that the quality of each material obtains with the ratio of its molar mass, the ratio namely obtaining the amount of substance of each monose in the raspberry folic acid polysaccharide (L-Ps-1) that embodiment 5 prepares is: rhamnosyl: pectinose: wood sugar: glucose: semi-lactosi=2.47:4.75:4.12:1:2.48.In visible L-Ps-1, the content of pectinose and wood sugar is higher, be respectively 32.05% and 27.80%, and the content of glucose is minimum, only accounts for 6.74%.Embodiment 6 and 7 result is with embodiment 5.
Embodiment 9Griess method detects the impact of L-Ps on RAW264.7 cell release NO
Cellar culture cell, the RAW264.7 cell of taking the logarithm vegetative period, piping and druming cell becomes single cell suspension, with after blood cell counting plate counting under microscope, 1000rpm/min, centrifugal 5min removes supernatant, substratum is resuspended and adjust cell concn, be inoculated in 24 well culture plates by the cell density in 200,000/hole, be placed in 37 DEG C, 5%CO
2incubator is cultivated, adherent 24h, supernatant is abandoned in suction, by following grouping requirement administration, often group establishes 3 multiple holes: Control group, lipopolysaccharides (LPS, final concentration is 1 μ g/mL) group, LPS (final concentration is 1 μ g/mL)+dexamethasone (DXM, final concentration is 50 μ g/mL) group, LPS (final concentration is 1 μ g/mL)+L-Ps (the leaf of Palmleaf Raspberry Crude polysaccharides that embodiment 1 prepares, final concentration is 2,20,200,400 μ g/mL) group, control group adds the substratum of same volume.After administration, be placed in 37 DEG C, 5%CO
2incubator, continues to cultivate 24h.Get each porocyte supernatant 100 μ L respectively, correspondingly to add in another 96 new orifice plates.Every hole adds 50 μ L Griess reagent A, be placed in 37 DEG C of incubators and react 10min, every hole adds 50 μ L Griess reagent B again, is placed in 37 DEG C of incubators and reacts 10min, put plate immediately on multiple labeling microwell plate detector, under 550nm wavelength, detect each hole absorbance.
The release of the RAW264.7 scavenger cell NO that L-Ps can suppress LPS to induce, and present gradient dependency (Fig. 5).
The impact that RAW264.7 cell TNF-α, iNOS and IL-6mRNA that embodiment 10L-Ps induces LPS express
Cellar culture cell, gets the cell to the growth number phase, is inoculated in 6 orifice plates by 1,000,000/porocyte number, after hatching 24h, inhales and abandons supernatant, and by following grouping requirement administration, often group establishes 3 multiple holes.Control group, lipopolysaccharides (LPS, final concentration is 1 μ g/mL) group, LPS (final concentration is 1 μ g/mL)+dexamethasone (DXM, final concentration is 50 μ g/mL) group, LPS (final concentration is 1 μ g/mL)+L-Ps (the leaf of Palmleaf Raspberry Crude polysaccharides that embodiment 1 prepares, final concentration is 5,25,50,100 μ g/mL), control group adds the substratum of same volume.After administration, be placed in 37 DEG C, 5%CO
2incubator, continues to cultivate 12h.After effect 12h, inhale and abandon cell conditioned medium, PBS washes 2 times.Every hole adds Trizol 1mL, leave standstill 5min, suction pipe is blown and beaten to liquid without dope, draw cell pyrolysis liquid and proceed to EP pipe, often pipe adds 0.2mL chloroform, cover EP pipe lid, hand-heldly firmly shake 10s up and down, room temperature leaves standstill 5min, 4 DEG C with 12, the centrifugal 15min of 000rpm, centrifugal rear transfer upper strata aqueous phase is in another EP pipe, add 0.5mL Virahol, room temperature leaves standstill 10min, 4 DEG C of whizzers are with 12, the centrifugal 10min of 000rpm, careful suction supernatant discarded, 2 times are cleaned with cold 75% ethanol 1mL, with 7 under difference 4 DEG C of conditions, the centrifugal 5min of 500rpm, supernatant is abandoned in careful suction, air blow drying, about 15min, add 0 ~ 50 μ L RNase-free pure water, 60 DEG C of heating 10min dissolution precipitations.Measure purity and the concentration of mRNA.And with RevertAid First Strand cnthesis Kit (Thermo company) Reverse Transcription box, 20 μ L reaction systems, carry out reverse transcription to RNA.Adopt DyNAmo Flash SYRB Green qPCR Kit (Thermo company) test kit, ABI real-time fluorescence quantitative PCR instrument (Rrism 7500, Applied Biosystems, FosterCity, CA, USA) mRNA is increased, after amplification, carry out automatic analysis to obtain the relative expression quantity of goal gene by RelativeQuantification (ddCt) Study method in ABI PRISM 7500SDS software.Related gene mRNA primer sequence is TNF-α (forward, 5 '-GGG GAT TAT GGC TCA GGG TC-3 ', reverse, 5 '-CGA GGC TCC AGT GAA TTC GG-3 '), IL-6 (forward, 5 '-GTA CTCCAG AAG ACC AGA GG-3 ', reverse, 5 '-TGC TGG TGA CAA CCA CGG CC-3 '), iNOS (forward, 5 '-CGG CAA ACA TGA CTT CAG GC-3 ', reverse, 5 '-GCA CATCAA AGC GGC CAT AG-3 ') and GAPDH (forward, 5 '-CAC TCA CGG CAA ATTCAA CGG CAC-3 ', reverse, 5 '-GAC TCC ACG ACA TAC TCA GCA-3 ').
Compared with control group, TNF-α, iNOS and IL-6 that LPS component is secreted significantly increase (P < 0.01), show that LPS induces the inflammatory model of RAW264.7 scavenger cell release NO to set up.L-Ps significantly can suppress the secretion (P < 0.05) of TNF-α, iNOS and IL-6, and presents gradient dependency (Fig. 6 ~ 8).
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. a preparation method for leaf of Palmleaf Raspberry Crude polysaccharides, is characterized in that comprising following steps:
(1) by raspberry leaf grinding and sieving, leaf of Palmleaf Raspberry powder is obtained; 75 DEG C ~ 95 DEG C hot water returns extract Rubus chingii leaf powder, then that extracting solution is centrifugal, get supernatant liquid, filter 2 ~ 4 times, by filtrate reduced in volume, obtain polysaccharide soln;
(2) mix by polysaccharide soln with through pretreated macroporous adsorbent resin, decolour in 40 DEG C ~ 60 DEG C water-bath 2 ~ 4h, then filter out polysaccharide soln, and the polysaccharide adsorbed in resin is repeatedly washed out with water, merged by polysaccharide soln after decolouring, concentrating under reduced pressure, obtains concentrated liquid glucose;
(3) ethanol adding 3 ~ 5 times of volumes in concentrated liquid glucose carries out alcohol precipitation, then centrifugal, abandoning supernatant, and collecting precipitation thing is also dry, obtains leaf of Palmleaf Raspberry Crude polysaccharides.
2. the preparation method of leaf of Palmleaf Raspberry Crude polysaccharides according to claim 1, is characterized in that:
The ratio of water to material that hot water return described in step (1) extracts is (10:1) ~ (30:1);
The time that hot water return described in step (1) extracts is 2 ~ 4h, and the number of times that hot water return extracts is 2 ~ 4 times.
3. the preparation method of leaf of Palmleaf Raspberry Crude polysaccharides according to claim 1, is characterized in that:
Macroporous adsorbent resin described in step (2) is D354FD resin;
The pretreated method of the macroporous adsorbent resin described in step (2) is: be first soaked in water macroporous adsorbent resin 12 ~ 24h, then successively with massfraction be 5% hydrochloric acid soln and massfraction be 5% sodium hydroxide solution soak 0.5 ~ 3h separately, filter, be washed to neutrality, obtain through pretreated macroporous adsorbent resin.
4. the preparation method of leaf of Palmleaf Raspberry Crude polysaccharides according to claim 1, is characterized in that:
The temperature of step (1) and the concentrating under reduced pressure described in (2) is 40 ~ 60 DEG C;
The temperature of the alcohol precipitation described in step (3) is 0 ~ 4 DEG C, and the time of alcohol precipitation is 8 ~ 24h.
5. the preparation method of leaf of Palmleaf Raspberry Crude polysaccharides according to claim 1, is characterized in that:
Centrifugal rotating speed described in step (1) is 3000 ~ 5000rpm/min; The described centrifugal time is 8 ~ 12min;
Centrifugal rotating speed described in step (3) is 3000 ~ 5000rpm/min; The described centrifugal time is 12 ~ 15min.
6. a leaf of Palmleaf Raspberry Crude polysaccharides, is characterized in that being prepared by the preparation method described in any one of Claims 1 to 5.
7. a raspberry folic acid polysaccharide, is characterized in that obtaining by being further purified by leaf of Palmleaf Raspberry Crude polysaccharides according to claim 6;
Described raspberry folic acid polysaccharide is homogeneous components, and its molecular weight is 12686Da;
Described raspberry folic acid polysaccharide is the mixed polysaccharide comprising five kinds of monose, and wherein, saccharide residue mol ratio is: rhamnosyl: pectinose: wood sugar: glucose: semi-lactosi=2.47:4.75:4.12:1:2.48.
8. raspberry folic acid polysaccharide according to claim 7, is characterized in that:
Described raspberry folic acid polysaccharide prepares as follows:
(1) will proceed to after 40 ~ 60 DEG C of rotary evaporation bubble removings in Z-type chromatography column through pretreated DEAE-52 resin, even to make filler fill post with constant flow pump pumping distilled water, after balance, flow velocity 5 ~ 10s/ drips;
(2) leaf of Palmleaf Raspberry Crude polysaccharides is mixed with polysaccharide soln, proceed in chromatography column, use distilled water, 0.05mol/L NaCl, 0.1mol/L NaCl, 0.3mol/L NaCl, 0.5mol/L NaCl solution wash-out successively, flow velocity 5 ~ 10s/ drips, collect each stream part respectively, phend-sulphuric acid tracing detection elutriant, measure the absorbancy under 490nm, draw elution curve, the elutriant in same absorption peak merges; Then concentrated by rotary evaporation, vacuum lyophilization, obtains three components after DEAE-52 ion-exchange chromatography, and wherein, the component that content is the highest is raspberry folic acid polysaccharide.
9. raspberry folic acid polysaccharide according to claim 8, is characterized in that:
The pretreated method of DEAE-52 resin described in step (1) is: 4 ~ 30 DEG C, DEAE-52 resin water is soaked 12 ~ 24h; 0.5 ~ 2h is soaked again with the hydrochloric acid soln of 0.5mol/L; The NaOH solution of 0.5mol/L soaks 0.5 ~ 2h, filters, is washed to neutrality, obtains through pretreated DEAE-52 resin.
10. leaf of Palmleaf Raspberry Crude polysaccharides according to claim 6 or the raspberry folic acid polysaccharide described in any one of claim 7 ~ 9 are preparing the application in anti-inflammatory drug.
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