CN104829737B - A kind of palmleaf raspberry leaf Thick many candies and preparation method and application - Google Patents
A kind of palmleaf raspberry leaf Thick many candies and preparation method and application Download PDFInfo
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Abstract
The invention belongs to the field of Chinese medicines, and in particular to a kind of palmleaf raspberry leaf Thick many candies and preparation method and application.The steps such as the present invention is extracted by hot water return, decolorization and alcohol precipitation, palmleaf raspberry leaf Thick many candies are extracted from the leaf of raspberry, the Thick many candies can significantly inhibit the RAW264.7 macrophages NO of LPS inductions release in vitro, and can significantly inhibit RAW264.7 macrophage TNF α, iNOS and IL 6mRNA of LPS inductions expression.In addition, the present invention is further purified by ion-exchange chromatography method to palmleaf raspberry leaf Thick many candies, three components are obtained, wherein content highest component is raspberry folic acid polysaccharide, and the smart polysaccharide is the heteroglycan for including five kinds of monose, wherein, saccharide residue mol ratio is：Rhamnose：Arabinose：Xylose：Glucose：Galactolipin=2.47:4.75:4.12:1:2.48.
Description
Technical field
The invention belongs to the field of Chinese medicines, and in particular to a kind of palmleaf raspberry leaf Thick many candies and preparation method and application.
Background technology
To be that use can strengthen host defense anti-for one of most promising Antiinflammatory Regimen for substituting classical antibiotic therapy at present
The immunomodulator answered.Existing known panimmunity conditioning agent, including mammalian proteins, such as：Gamma interferon, granulocyte collection
G-CSF and granulocyte-macrophage colony stimutaing factor and the material isolated and purified from microorganism.For bacterium is immunized
For polysaccharide and artificial synthesized compound, its adverse reaction and side effect have caused people to note extensively.Most of high plants
The polysaccharide in thing source is the material having no adverse reaction, and larger side effect will not be produced to body, therefore, is separated from plant
Polysaccharide caused great concern in biomedicine.The mechanism of action of these active materials is also evolving, wherein polysaccharide
The immunologic mechanism of non-specificity induction is more valued by people.Research finds that plant polyose is antitumor, sterilize and other are controlled
The immune activation mechanism for the treatment of effect is completed by the regulation of complement system and the stimulation of macrophage.Research discovery, plant
The polysaccharide in source can embody various beneficial pharmacological actions by adjusting the immunologic function of macrophage.Plant polyose is anti-
Optimal vaccine candidate medicine that is scorching, antitumor and promoting wound healing.
Raspberry (Rubus chingii Hu), i.e. Rubus chingii, are the rose family (Rosaceae) rubus,
Main product is saved in Anhui, Jiangxi, Jiangsu etc. in Zhejiang, Fujian, in addition and is also distributed, and is covered because it is distributed mainly on East China and is also known as East China
Basin.Raspberry is《Pharmacopoeia of People's Republic of China》Version one is included within 2010, using its prematurity dry fruit as medicinal portion
Position, the effect of with kidney tonifying, controlling nocturnal emission with astringent drugs, contracting urine, for enuresis with renal asthenia, frequent urination, positive paralysis premature ejaculation, emission and spermatorrhea etc..For covering
The research of basin fruit polysaccharide is more, but the research for raspberry leaf polysaccharide does not have but.
The content of the invention
In order to overcome the deficiencies in the prior art and shortcoming, primary and foremost purpose of the invention is to provide a kind of palmleaf raspberry leaf slightly many
The preparation method of sugar.
Another object of the present invention is to provide the palmleaf raspberry leaf Thick many candies that above-mentioned preparation method is prepared.
It is still another object of the present invention to provide the application of above-mentioned palmleaf raspberry leaf Thick many candies.
The purpose of the present invention is realized by following proposal：
A kind of preparation method of palmleaf raspberry leaf Thick many candies, is comprised the following steps：
(1) it will be sieved after raspberry (Rubus chingii Hu) crushing up leaves, obtain palmleaf raspberry leaf powder；75 DEG C~95 DEG C
Hot water return extracts Rubus chingii leaf powder, then centrifuges extract solution, takes supernatant liquid, filters 2~4 times, filtrate is subtracted
Pressure concentration, obtains polysaccharide solution；
(2) macroporous absorbent resin by polysaccharide solution and by pretreatment is mixed, and is carried out in 40 DEG C~60 DEG C 2~4h of water-bath
Decolourize, then filter out polysaccharide solution, and wash out the polysaccharide adsorbed in resin repeatedly with water, the polysaccharide solution after decolouring is closed
And, it is concentrated under reduced pressure, obtains concentrating liquid glucose；
(3) ethanol that 3~5 times of volumes are added in concentration liquid glucose carries out alcohol precipitation, is then centrifuged for, abandoning supernatant, collects
Sediment and drying, obtain palmleaf raspberry leaf Thick many candies (L-Ps)；
The ratio of water to material that hot water return described in step (1) is extracted is (10:1)~(30:1)；
The time that hot water return described in step (1) is extracted is 2~4h, and the number of times that hot water return is extracted is 2~4 times；
The rotating speed of centrifugation described in step (1) is 3000~5000rpm/min；The time of described centrifugation be 8~
12min；
Macroporous absorbent resin described in step (2) is preferably D354FD resins；
The method of the pretreatment of macroporous absorbent resin described in step (2) is：Macroporous absorbent resin is first soaked in water
12~24h, the sodium hydroxide solution that the hydrochloric acid solution and mass fraction for being successively then 5% with mass fraction are 5% each soaks
0.5~3h is steeped, filtering is washed to neutrality, obtains the macroporous absorbent resin by pretreatment；
The temperature being concentrated under reduced pressure described in step (1) and (2) is 40~60 DEG C；
The volume fraction of ethanol described in step (3) is 70%~100%；
The temperature of alcohol precipitation described in step (3) is 0~4 DEG C, and the time of alcohol precipitation is 8~24h；
The rotating speed of centrifugation described in step (3) is 3000~5000rpm/min；The time of described centrifugation be 12~
15min；
The temperature of drying described in step (3) is 40~60 DEG C；
A kind of palmleaf raspberry leaf Thick many candies (L-Ps), are prepared by above-mentioned preparation method；
A kind of raspberry folic acid polysaccharide (L-Ps-1), is obtained by the way that above-mentioned palmleaf raspberry leaf Thick many candies are further purified；
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 the heteroglycan for including five kinds of monose, wherein, saccharide residue mol ratio
For：Rhamnose：Arabinose：Xylose：Glucose：Galactolipin=2.47:4.75:4.12:1:2.48；
Described raspberry folic acid polysaccharide (L-Ps-1), which is made by the steps, to be obtained：
(1) by the DEAE-52 resins by pretreatment in being transferred to Z-type chromatographic column after 40~60 DEG C of rotary evaporation bubble removings
In, with constant flow pump pumping distilled water so that filler fills post uniformly, 5~10s/ of flow velocity drops after balance；
(2) palmleaf raspberry leaf Thick many candies are configured to polysaccharide solution, be transferred in chromatographic column, successively with distilled water, 0.05mol/L
NaCl, 0.1mol/L NaCl, 0.3mol/L NaCl, 0.5mol/L NaCl solution are eluted, and flow velocity 5~10s/ drops are collected respectively
Each stream part, phend-sulphuric acid tracing detection eluent determines the absorbance under 490nm, draws elution curve, same absworption peak
Interior eluent merges；Then concentrated by rotary evaporation, vacuum freeze drying, obtain three after DEAE-52 ion-exchange chromatographies
Component, wherein, content highest component is raspberry folic acid polysaccharide (L-Ps-1)；
Described in step (1) DEAE-52 resins pretreatment method be：By 4~30 DEG C of immersions of water of DEAE-52 resins
12~24h；Again 0.5~2h is soaked with 0.5mol/L hydrochloric acid solution；0.5mol/L NaOH solution soaks 0.5~2h, filters,
Neutrality is washed to, the DEAE-52 resins by pretreatment are obtained；
The temperature of concentrated by rotary evaporation described in step (2) is 40~60 DEG C；
Application of the described palmleaf raspberry leaf Thick many candies or raspberry folic acid polysaccharide in anti-inflammatory drug is prepared；
Described palmleaf raspberry leaf Thick many candies or raspberry folic acid polysaccharide can be as new anti-inflammatory drugs, applied to inflammation
Treatment.
A kind of anti-inflammatory drug, contains above-mentioned palmleaf raspberry leaf Thick many candies or raspberry folic acid polysaccharide；
The principle of the present invention：The polysaccharide of plant origin can by adjust the immunologic function of macrophage embody it is various beneficial
Pharmacological action.Present invention discover that and confirmation plant polyose L-Ps there is significant anti-inflammatory activity, and plant polyose L-Ps makes
Low with dosage, the toxic side effect under same dosage is relatively low.
The present invention has the following advantages and effect relative to prior art：
(1) present invention firstly discovers that palmleaf raspberry leaf Thick many candies can significantly inhibit the RAW264.7 macrophages of LPS inductions in vitro
Cell NO release.
(2) present invention firstly discovers that palmleaf raspberry leaf Thick many candies can significantly inhibit the RAW264.7 macrophages of LPS inductions in vitro
The expression of cell TNF-α, iNOS and IL-6mRNA.
(3) dosage of palmleaf raspberry leaf Thick many candies is than relatively low, and toxicity is relatively low in the range of effective dosage.
Brief description of the drawings
Fig. 1 is the ultraviolet scanning spectrum figure for the palmleaf raspberry leaf Thick many candies that embodiment 1 is prepared.
Fig. 2 is the infrared spectrogram for the palmleaf raspberry leaf Thick many candies that embodiment 1 is prepared.
Fig. 3 is the ion-exchange chromatography elution curve of embodiment 5.
Fig. 4 is the GPC figures for the raspberry folic acid polysaccharide that embodiment 5 is prepared.
Fig. 5 is the influence figure that the RAW264.7 macrophages that palmleaf raspberry leaf Thick many candies are induced LPS discharge NO.
Fig. 6 is the influence figure for the RAW264.7 macrophage TNF-αs mRNA expression that palmleaf raspberry leaf Thick many candies are induced LPS,
Wherein, ##：Compared with control groups, P<0.01.
Fig. 7 is the influence figure for the RAW264.7 macrophage iNOS mRNA expression that palmleaf raspberry leaf Thick many candies are induced LPS,
Wherein, ##：Compared with control groups, P<0.01.
Fig. 8 is the influence figure for the RAW264.7 macrophages IL-6mRNA expression that palmleaf raspberry leaf Thick many candies are induced LPS, its
In, ##：Compared with control groups, P<0.01.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited
In this.
In embodiment, mouse macrophage RAW 264.7 is purchased from Shanghai life science institute of Chinese Academy of Sciences cellular resources
The heart；Rubus chingii leaf picks up from Zhejiang farmers'.
Embodiment 1 prepares palmleaf raspberry leaf Thick many candies (L-Ps) from Rubus chingii leaf
(1) 20 mesh sieves are crossed after Rubus chingii crushing up leaves, 100g is weighed, uses distilled water heating and refluxing extraction, wherein, water
Material is than being 20:1, refluxing extraction temperature is 75 DEG C, and reflux extracting time is 4h, refluxing extraction number of times 3 times；It is then combined with extracting
Centrifuge 10min under liquid, 4000rpm/min, take supernatant liquid, filter paper filter 23, by filtrate with Rotary Evaporators at 40 DEG C
200mL is concentrated under reduced pressure into, polysaccharide solution is obtained；
(2) D354FD resins are first used into distilled water immersion 12h, hydrochloric acid solution then successively with mass fraction for 5%, matter
Amount fraction each soaks 0.5h for 5% sodium hydroxide solution, and 200 mesh filtered through gauze, distillation is washed to neutrality, obtained by pre-
The D354FD resins of processing；
(3) D354FD mixed with resin of the polysaccharide solution and 200mL prepared step (1) by pretreatment, is placed in
Water bath with thermostatic control 3h is decolourized in 50 DEG C of thermostat water baths, interval stirring, then goes out polysaccharide solution with the filter-cloth filtering of 200 mesh,
And wash out the polysaccharide adsorbed in resin repeatedly with water, the polysaccharide liquid after decolouring is merged, depressurized with Rotary Evaporators at 40 DEG C
200mL is concentrated into, obtains concentrating liquid glucose；
(4) ethanol that the volume fraction of 4 times of volumes is 95%, side are added in the concentration liquid glucose that step (3) is prepared
Then edged magnetic agitation, the alcohol precipitation 8h under the conditions of 4 DEG C centrifuges 15min under 4000rpm/min, abandoning supernatant is taken out
Sediment obtains palmleaf raspberry leaf Thick many candies (L-Ps) in 45 DEG C of drying.
Embodiment 2 prepares palmleaf raspberry leaf Thick many candies (L-Ps) from Rubus chingii leaf
(1) 20 mesh sieves are crossed after Rubus chingii crushing up leaves, 100g are weighed, 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；It is then combined with extracting
Centrifuge 8min under liquid, 3000rpm/min, take supernatant liquid, filter paper is filtered 2 times, by filtrate with Rotary Evaporators at 50 DEG C
200mL is concentrated under reduced pressure into, polysaccharide solution is obtained；
(2) D354FD resins are first used into distilled water immersion 24h, hydrochloric acid solution then successively with mass fraction for 5%, matter
Amount fraction each soaks 2h for 5% sodium hydroxide solution, and 200 mesh filtered through gauze, distillation is washed to neutrality, obtains by pre- place
The D354FD resins of reason；
(3) D354FD mixed with resin of the polysaccharide solution and 200mL prepared step (1) by pretreatment, is placed in
Water bath with thermostatic control 2h is decolourized in 40 DEG C of thermostat water baths, interval stirring, then goes out polysaccharide solution with the filter-cloth filtering of 200 mesh,
And wash out the polysaccharide adsorbed in resin repeatedly with water, the polysaccharide liquid after decolouring is merged, depressurized with Rotary Evaporators at 50 DEG C
200mL is concentrated into, obtains concentrating liquid glucose；
(4) ethanol that the volume fraction of 3 times of volumes is 95% is added in the concentration liquid glucose prepared in step (3),
Then side edged magnetic agitation, the alcohol precipitation 12h under the conditions of 0 DEG C centrifuges 12min under 3000rpm/min, abandoning supernatant,
Sediment is taken out in 50 DEG C of drying, palmleaf raspberry leaf Thick many candies (L-Ps) are obtained.
Embodiment 3 prepares palmleaf raspberry leaf Thick many candies (L-Ps) from Rubus chingii leaf
(1) 20 mesh sieves are crossed after Rubus chingii crushing up leaves, 100g is weighed, uses distilled water heating and refluxing extraction, wherein, water
Material is than being 20:1, refluxing extraction temperature is 95 DEG C, and reflux extracting time is 2h, and refluxing extraction number of times 4 times is then combined with extracting
Centrifuge 12min under liquid, 5000rmp/min, take supernatant liquid, filter paper is filtered 4 times, by filtrate with Rotary Evaporators at 60 DEG C
200mL is concentrated under reduced pressure into, polysaccharide solution is obtained；
(2) D354FD resins are first used into distilled water immersion 18h, hydrochloric acid solution then successively with mass fraction for 5%, matter
Amount fraction each soaks 3h for 5% sodium hydroxide solution, and 200 mesh filtered through gauze, distillation is washed to neutrality, obtains by pre- place
The D354FD resins of reason；
(3) the D354FD mixed with resin by the polysaccharide solution and 200mL that are prepared in (1) by pretreatment, is placed in 60
Water bath with thermostatic control 4h is decolourized in DEG C thermostat water bath, interval stirring, goes out polysaccharide solution with the filter-cloth filtering of 200 mesh afterwards, and
Wash out the polysaccharide adsorbed in resin repeatedly with water, the polysaccharide liquid after decolouring is merged, depressurized with Rotary Evaporators at 60 DEG C dense
200mL is reduced to, obtains concentrating liquid glucose；
(4) ethanol that the volume fraction of 5 times of volumes is 95%, side are added in the concentration liquid glucose that step (3) is prepared
Then edged magnetic agitation, the alcohol precipitation 24h under the conditions of 4 DEG C centrifuges 14min under 5000rpm/min, abandoning supernatant takes
Go out sediment in 60 DEG C of drying, obtain palmleaf raspberry leaf Thick many candies (L-Ps).
The UV scanning collection of illustrative plates of embodiment 4 and IR spectrum scanning
(1) palmleaf raspberry leaf Thick many candies (L-Ps) 4mg prepared respectively in Example 1~3, is configured to 1mg/mL's
Polysaccharide solution, using distilled water as control, surveys its absorbance at 190~500nm.
(2) palmleaf raspberry leaf Thick many candies (L-Ps) 2mg prepared respectively in Example 1~3, mixes finely ground with KBr
Into thin uniform layer shape, in 4000~400cm-1Infrared scan is carried out in wave-number range.
Interpretation of result：
(1) polysaccharide ultraviolet spectral analysis result：The palmleaf raspberry leaf Thick many candies prepared in embodiment 1 200~
280nm has stronger absorption, shows that unsaturated carbonyl and carboxyl may be contained in sample, there is stronger absworption peak at 266nm,
Show the nucleic acid or protein (Fig. 1) containing conjugation in sample.The testing result be the same as Example 1 of embodiment 2 and embodiment 3.
(2) polysaccharide results of IR：The palmleaf raspberry leaf Thick many candies prepared in embodiment 1 are in wave number
3345cm-1The strong absworption peak at place is between carbohydrate molecule or the O-H stretching vibration peaks of intramolecular, in wave number 2933cm-1What place was present
Absworption peak is the C-H vibration absorption peaks of methine or methyl, in 1430~1200cm of wave number-1The absworption peak at place is the C-O of carboxyl
Stretching vibration peak, the characteristic peak of this three groups of carbohydrates may determine that the palmleaf raspberry leaf Thick many candies prepared in embodiment 1 are polysaccharide.
In 1000~1200cm of wave number-1The absworption peak at place is the vibration peak of uronic acid, in wave number 1617cm-1The relatively weak suction at place
Receive the characteristic IR absorbance peaks (Fig. 2) that peak is protein.These results further illustrate the raspberry prepared in embodiment 1
Leaf Thick many candies contain polysaccharide, protein and uronic acid.The testing result be the same as Example 1 of embodiment 2 and embodiment 3.
The preparation of the raspberry folic acid polysaccharide (L-Ps-1) of embodiment 5
(1) DEAE-52 is pre-processed：70g DEAE-52 is taken, with 4 DEG C of immersion 24h of distilled water, is then carefully fallen upper water
Go out, remove impurity, then 0.5h is soaked with 0.5mol/L hydrochloric acid solution, upper strata acid solution is carefully poured out, suction filtration is to after doing with steaming
Distilled water is eluted to neutrality, then soaks 0.5h with 0.5mol/L NaOH solution, carefully pours out upper strata alkali lye, suction filtration is to after doing with steaming
Distilled water is washed till neutrality, standby；
(2) DEAE-52 by pretreatment is transferred to 2.6 × 30cm in glass bar drainage after 48 DEG C of rotary evaporation bubble removings
In the Z-type chromatographic column of specification, with constant flow pump pumping distilled water so that filler fills post uniformly, 7s/ drops after balance；
(3) the palmleaf raspberry leaf Thick many candies (L-Ps) prepared in 100mg embodiments 1 are weighed, polysaccharide solution, glass is configured to
Glass rod is drained in chromatographic column, successively with distilled water, 0.05mol/L NaCl, 0.1mol/L NaCl, 0.3mol/L NaCl,
0.5mol/L NaCl solutions are eluted, and each stream part is collected automatically, and every pipe collects about 5mL, and phend-sulphuric acid tracing detection is washed
De- liquid, determines the absorbance at 490nm, and the eluent drawn in elution curve, same absworption peak merges；Then 40 DEG C of eluent
Concentrated by rotary evaporation, vacuum freeze drying, wherein, L-Ps obtains three components after DEAE-52 ion-exchange chromatographies, and content is most
High component (48.0%) is raspberry folic acid polysaccharide (L-Ps-1), wherein, Fig. 3 is the ion-exchange chromatography of the present embodiment
Elution curve.
The preparation of the raspberry folic acid polysaccharide (L-Ps-1) of embodiment 6
(1) DEAE-52 is pre-processed：70g DEAE-52 is taken, it is then careful by upper water with 20 DEG C of immersion 12h of distilled water
Pour out, remove impurity, then 1h is soaked with 0.5mol/L hydrochloric acid solution, upper strata acid solution is carefully poured out, suction filtration is to after doing with steaming
Distilled water is eluted to neutrality, then soaks 1h with 0.5mol/L NaOH solution, carefully pours out upper strata alkali lye, suction filtration is to after doing with distillation
Neutrality is washed to, it is standby；
(2) DEAE-52 by pretreatment is transferred to 2.6 × 30cm in glass bar drainage after 48 DEG C of rotary evaporation bubble removings
In the Z-type chromatographic column of specification, with constant flow pump pumping distilled water so that filler fills post uniformly, 7s/ drops after balance；
(3) the palmleaf raspberry leaf Thick many candies (L-Ps) prepared in 100mg embodiments 1 are weighed, polysaccharide solution, glass is configured to
Glass rod is drained in chromatographic column, successively with distilled water, 0.05mol/L NaCl, 0.1mol/L NaCl, 0.3mol/L NaCl,
0.5mol/L NaCl solutions are eluted, and each stream part is collected automatically, and every pipe collects about 5mL, and phend-sulphuric acid tracing detection is washed
De- liquid, determines the absorbance at 490nm, and the eluent drawn in elution curve, same absworption peak merges；Then 50 DEG C of eluent
Concentrated by rotary evaporation, vacuum freeze drying, wherein, L-Ps obtains three components after DEAE-52 ion-exchange chromatographies, and content is most
High component (48.0%) is raspberry folic acid polysaccharide (L-Ps-1), wherein, the ion-exchange chromatography elution of the present embodiment
As a result be the same as Example 5.
The preparation of the raspberry folic acid polysaccharide (L-Ps-1) of embodiment 7
(1) DEAE-52 is pre-processed：70g DEAE-52 is taken, it is then careful by upper water with 25 DEG C of immersion 20h of distilled water
Pour out, remove impurity, then 0.8h is soaked with 0.5mol/L hydrochloric acid solution, upper strata acid solution is carefully poured out, suction filtration is used to after doing
Distilled water is eluted to neutrality, then soaks 0.8h with 0.5mol/L NaOH solution, carefully pours out upper strata alkali lye, and suction filtration is used to after doing
Distillation is washed to neutrality, standby；
(2) DEAE-52 by pretreatment is transferred to 2.6 × 30cm in glass bar drainage after 48 DEG C of rotary evaporation bubble removings
In the Z-type chromatographic column of specification, with constant flow pump pumping distilled water so that filler fills post uniformly, 7s/ drops after balance；
(3) the palmleaf raspberry leaf Thick many candies (L-Ps) prepared in 100mg embodiments 1 are weighed, polysaccharide solution, glass is configured to
Glass rod is drained in chromatographic column, successively with distilled water, 0.05mol/L NaCl, 0.1mol/L NaCl, 0.3mol/L NaCl,
0.5mol/L NaCl solutions are eluted, and each stream part is collected automatically, and every pipe collects about 5mL, and phend-sulphuric acid tracing detection is washed
De- liquid, determines the absorbance at 490nm, and the eluent drawn in the same absworption peak of elution curve merges；Then 60 DEG C of eluent
Concentrated by rotary evaporation, vacuum freeze drying,
Wherein, L-Ps obtains three components, content highest component after DEAE-52 ion-exchange chromatographies
(48.0%) it is raspberry folic acid polysaccharide (L-Ps-1), wherein, the ion-exchange chromatography elution result of the present embodiment is with real
Apply example 5.
Embodiment 8L-Ps-1 molecular weight, Purity and monosaccharide composition analysis
(1) the raspberry folic acid polysaccharide (L-Ps-1) prepared respectively to embodiment 5~7 using high performance liquid chromatograph
Molecular weight and its purity be measured.Chromatographic condition：TSK G-5000PXL column (7.8 × 300mm) and TSK G-
3000PXL column (7.8 × 300mm) connect, and mobile phase is 0.02mol/L KH2PO4Cushioning liquid, flow velocity is 0.6mL/
Min, 2414 Composition distributions, 35 DEG C of column temperature.
(2) raspberry folic acid polysaccharide (L-Ps-1) 10mg that embodiment 5~7 prepares is weighed respectively is placed in 5mL amperes
Guan Zhong, adds 2mol/L trifluoroacetic acid solution 4mL, alcohol blast burner sealing, 110 DEG C of hydrolysis 6h, afterwards by 48 DEG C of hydrolyzation sample
It is concentrated under reduced pressure and is evaporated, add appropriate volume methanol, then 48 DEG C are concentrated under reduced pressure and are evaporated, is so repeated 3 times to sample in untill neutral.
10mg glucose, fucose, rhamnose, xylose, mannose, arabinose, gala saccharide and each hydrolysate are taken respectively
In 10mL screw socket pipes, 10mg hydroxylamine hydrochlorides, 1mL pyridines are added, in being reacted under 90 DEG C of water bath conditions in 30min, course of reaction
Every 5min vibrations once, addition acetic anhydride 1mL after room temperature is naturally cooled to, continues to react under 90 DEG C of water bath conditions
In 30min, course of reaction every 5min vibrations once, room temperature is naturally cooled to after taking-up, is ultimately generated with volatile sugar
Nitrile acetic ester derivative, crosses (0.22 μm) processing of film, GC analyses is directly carried out afterwards, according to the retention time of sample and standard items
Control, obtains the monose composition of various sample polysaccharide, and calculates various monose percentage by weights according to the area ratio at each peak.Color
Spectral condition：Aglient 6890N gas chromatographs；DB-1701 capillary columns (30m × 0.25m, 0.25 μm)；Temperature of vaporization chamber
250 DEG C, 300 DEG C of detector temperature, 180 DEG C of column temperature；Temperature programming：180 (2min) are warming up to 220 with the speed in 2 DEG C/min
DEG C, keep in 1min, be then warming up to 250 DEG C again with the speed in 5 DEG C/min, and keep in 2min.Detector：FID is detected
Device；Flow rate of carrier gas：1mL/min；Purging：4.0mL/min.
Interpretation of result：
(1) the GPC figures for the L-Ps-1 that embodiment 5 is prepared are single symmetrical peak, and it is homogeneous polysaccharide to show L-Ps-1, its
The equal molecular mass (Mn) of number is 12686Da, and weight average molecular mass (Mw) is 16073Da, and peak position molecular mass (Mp) is 17415Da
(Fig. 4).The result be the same as Example 5 of embodiment 6 and 7.
(2) according to area normalization method, the ratio between peak area is equal to contained substance mass ratio, according to the quality of each material with
What the ratio between its molal weight was obtained is the amount of the material of each material, that is, obtains the raspberry folic acid that embodiment 5 prepares many
The ratio between amount of material of each monose is in sugared (L-Ps-1)：Rhamnose：Arabinose：Xylose：Glucose：Galactolipin=2.47:
4.75:4.12:1:2.48.It can be seen that the content of arabinose and xylose is higher in L-Ps-1, respectively 32.05% and 27.80%,
And the content of glucose is minimum, 6.74% is only accounted for.The result be the same as Example 5 of embodiment 6 and 7.
Embodiment 9Griess methods detect that L-Ps discharges NO influence to RAW264.7 cells
Cellar culture cell, the RAW264.7 cells in growth period of taking the logarithm, piping and druming cell is into single cell suspension, under microscope
After being counted with blood cell counting plate, 1000rpm/min, centrifugation 5min removes supernatant, and culture medium is resuspended and adjusts cell concentration, by 20
The cell density in ten thousand/hole is inoculated in 24 well culture plates, is placed in 37 DEG C, 5%CO2Supernatant is abandoned in incubator culture, adherent 24h, suction,
By following packet requirement administration, every group sets 3 multiple holes：Control groups, lipopolysaccharides (LPS, final concentration of 1 μ g/mL) group, LPS
(final concentration of 1 μ g/mL)+dexamethasone (DXM, final concentration of 50 μ g/mL) group, LPS (final concentration of 1 μ g/mL)+L-Ps is (real
Apply the palmleaf raspberry leaf Thick many candies that example 1 is prepared, final concentration of 2,20,200,400 μ g/mL) group, control group addition same volume
Culture medium.After administration, 37 DEG C, 5%CO are placed in2Incubator, continues to cultivate 24h.Each μ L of hole cell conditioned medium 100 are taken respectively, accordingly
Add in another 96 new orifice plates.Add 50 μ L Griess reagent As per hole, be placed in 37 DEG C of incubators and react 10min, per hole again
Plus 50 μ L Griess reagent B, it is placed in 37 DEG C of incubators and reacts 10min, plate is put immediately on multiple labeling micropore board detector,
Each hole absorbance is detected under 550nm wavelength.
L-Ps can suppress the RAW264.7 macrophages NO of LPS inductions release, and gradient dependence (Fig. 5) is presented.
The influence of RAW264.7 cells TNF-α, iNOS and IL-6mRNA expression that embodiment 10L-Ps is induced LPS
Cellar culture cell, takes the cell to growing the number phase, and 6 orifice plates are inoculated in by 1,000,000/hole cell number, is incubated 24h
Afterwards, inhale and abandon supernatant, by following packet requirement administration, every group sets 3 multiple holes.Control groups, lipopolysaccharides (LPS, final concentration of 1 μ
G/mL) group, LPS (final concentration of 1 μ g/mL)+dexamethasone (DXM, final concentration of 50 μ g/mL) group, LPS (final concentration of 1 μ g/
ML)+L-Ps (the palmleaf raspberry leaf Thick many candies that embodiment 1 is prepared, final concentration of 5,25,50,100 μ g/mL), control group addition
The culture medium of same volume.After administration, 37 DEG C, 5%CO are placed in2Incubator, continues to cultivate 12h.Act on after 12h, suction is abandoned on cell
Clearly, PBS is washed 2 times.Trizol 1mL are added per hole, 5min is stood, suction pipe is blown and beaten to liquid without dope, draws cell pyrolysis liquid
Be transferred to EP pipes, often pipe adds 0.2mL chloroforms, covers EP lids, hand-held firmly concussion 10s up and down is stored at room temperature 5min, 4 DEG C with
12,000rpm centrifugation 15min, shift upper strata aqueous phase into another EP pipes, add 0.5mL isopropanols, be stored at room temperature after centrifugation
10min, 4 DEG C of centrifuges centrifuge 10min with 12,000rpm, carefully inhale supernatant discarding, are cleaned 2 times with cold 75% ethanol 1mL, point
5min is centrifuged with 7,500rpm under the conditions of other 4 DEG C, careful inhale abandons supernatant, and air blow drying, about 15min add 0~50 μ L
RNase-free pure water, 60 DEG C of heating 10min dissolving precipitations.Determine mRNA purity and concentration.And use RevertAid First
Strand cnthesis Kit (Thermo companies) reverse transcription reagent box, 20 μ L reaction systems carry out reverse transcription to RNA.Using
DyNAmo Flash SYRB Green qPCR Kit (Thermo companies) kit, ABI real-time fluorescence quantitative PCR instrument (Rrism
7500, Applied Biosystems, Foster City, CA, USA) mRNA is expanded, ABI PRISM are used after amplification
Relative Quantification (ddCt) Study methods are automatically analyzed to obtain target gene in 7500SDS softwares
Relative expression quantity.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 CTC CAG 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 CAT CAA AGC GGC CAT AG-3’)and GAPDH
(forward,5’-CAC TCA CGG CAA ATT CAA CGG CAC-3’,reverse,5’-GAC TCC ACG ACA TAC
TCA GCA-3’)。
Compared with control groups, TNF-α, iNOS and the IL-6 that LPS components are secreted dramatically increase (P ＜ 0.01), show LPS
RAW264.7 macrophages release NO inflammatory model is induced to set up.L-Ps can significantly inhibit TNF-α, iNOS and IL-6 point
Secrete (P ＜ 0.05), and gradient dependence (Fig. 6~8) is presented.
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (9)
1. a kind of palmleaf raspberry leaf Thick many candies, it is characterised in that be prepared via a method which to obtain：
(1) it will be sieved after Rubus chingii crushing up leaves, obtain Rubus chingii leaf powder；75 DEG C~95 DEG C hot water returns extract the palm
Leaf palmleaf raspberry leaf powder, then centrifuges extract solution, takes supernatant liquid, filters 2~4 times, filtrate decompression is concentrated, polysaccharide is obtained
Solution；
(2) macroporous absorbent resin by polysaccharide solution and by pretreatment is mixed, and is taken off in 40 DEG C~60 DEG C 2~4h of water-bath
Color, then filters out polysaccharide solution, and washes out the polysaccharide adsorbed in resin repeatedly with water, and the polysaccharide solution after decolouring is merged,
It is concentrated under reduced pressure, obtains concentrating liquid glucose；
(3) ethanol that 3~5 times of volumes are added in concentration liquid glucose carries out alcohol precipitation, is then centrifuged for, abandoning supernatant, collects precipitation
Thing and drying, obtain palmleaf raspberry leaf Thick many candies.
2. palmleaf raspberry leaf Thick many candies according to claim 1, it is characterised in that：
The ratio of water to material that hot water return described in step (1) is extracted is (10:1)~(30:1)；
The time that hot water return described in step (1) is extracted is 2~4h, and the number of times that hot water return is extracted is 2~4 times.
3. palmleaf raspberry leaf Thick many candies according to claim 1, it is characterised in that：
Macroporous absorbent resin described in step (2) is D354FD resins；
The method of the pretreatment of macroporous absorbent resin described in step (2) is：Macroporous absorbent resin is first soaked in water to 12~
24h, the sodium hydroxide solution that the hydrochloric acid solution and mass fraction for being successively then 5% with mass fraction are 5% each soaks 0.5
~3h, filtering, is washed to neutrality, obtains the macroporous absorbent resin by pretreatment.
4. palmleaf raspberry leaf Thick many candies according to claim 1, it is characterised in that：
The temperature being concentrated under reduced pressure described in step (1) and (2) is 40~60 DEG C；
The temperature of alcohol precipitation described in step (3) is 0~4 DEG C, and the time of alcohol precipitation is 8~24h.
5. palmleaf raspberry leaf Thick many candies according to claim 1, it is characterised in that：
The rotating speed of centrifugation described in step (1) is 3000~5000r/min；The time of described centrifugation is 8~12min；
The rotating speed of centrifugation described in step (3) is 3000~5000r/min；The time of described centrifugation is 12~15min.
6. a kind of raspberry folic acid polysaccharide, it is characterised in that by the way that the palmleaf raspberry leaf described in any one of Claims 1 to 5 is slightly more
Sugar, which is further purified, to be obtained；
Described raspberry folic acid polysaccharide is homogeneous components, and its molecular weight is 12686Da；
Described raspberry folic acid polysaccharide is the heteroglycan for including five kinds of monose, wherein, saccharide residue mol ratio is：Rhamnose：Ah
Draw uncle's sugar：Xylose：Glucose：Galactolipin=2.47:4.75:4.12:1:2.48.
7. raspberry folic acid polysaccharide according to claim 6, it is characterised in that：
Described raspberry folic acid polysaccharide, which is made by the steps, to be obtained：
(1) the DEAE-52 resins by pretreatment are used in being transferred to after 40~60 DEG C of rotary evaporation bubble removings in Z-type chromatographic column
Constant flow pump pumps distilled water so that filler dress post is uniform, 5~10s/ of flow velocity drops after balance；
(2) palmleaf raspberry leaf Thick many candies are configured to polysaccharide solution, be transferred in chromatographic column, successively with distilled water, 0.05mol/L
NaCl, 0.1mol/L NaCl, 0.3mol/L NaCl, 0.5mol/L NaCl solution are eluted, and flow velocity 5~10s/ drops are collected respectively
Each stream part, phend-sulphuric acid tracing detection eluent determines the absorbance under 490nm, draws elution curve, same absworption peak
Interior eluent merges；Then concentrated by rotary evaporation, vacuum freeze drying, obtain three after DEAE-52 ion-exchange chromatographies
Component, wherein, content highest component is raspberry folic acid polysaccharide.
8. raspberry folic acid polysaccharide according to claim 7, it is characterised in that：
Described in step (1) DEAE-52 resins pretreatment method be：By DEAE-52 resins with 4~30 DEG C of water immersion 12~
24h；Again 0.5~2h is soaked with 0.5mol/L hydrochloric acid solution；0.5mol/L NaOH solution soaks 0.5~2h, filters, washing
To neutral, the DEAE-52 resins by pretreatment are obtained.
9. the raspberry described in palmleaf raspberry leaf Thick many candies or any one of claim 6~8 described in any one of Claims 1 to 5
Application of the folic acid polysaccharide in anti-inflammatory drug is prepared.
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| CN107266603A (en) * | 2017-07-29 | 2017-10-20 | 安徽博格生物科技有限公司 | It is a kind of that there is the raspberry polysaccharide component extracting method for removing free radical function |
| CN114917246B (en) * | 2022-05-30 | 2023-09-26 | 华南师范大学 | Application of raspberry polysaccharide R2 in preparation of anti-tumor drugs and anti-inflammatory preparations |
| CN114917245B (en) * | 2022-05-30 | 2023-09-26 | 华南师范大学 | Application of raspberry polysaccharide R1 in preparation of anti-tumor drugs and anti-inflammatory preparations |
| CN114886088A (en) * | 2022-06-16 | 2022-08-12 | 果原力(北京)生物科技有限公司 | White kidney bean bubble water and preparation method thereof |
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