CN106478833A - A kind of Calyx Hibisci Sabdariffae polysaccharide and preparation method and application - Google Patents
A kind of Calyx Hibisci Sabdariffae polysaccharide and preparation method and application Download PDFInfo
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Abstract
The invention belongs to the technical field of Chinese medicine, disclose a kind of Calyx Hibisci Sabdariffae polysaccharide and preparation method and application.The present invention is by steps such as hot water return extraction, desolventing technology and precipitate with ethanol, Calyx Hibisci Sabdariffae crude polysaccharides are extracted from the calyx of Calyx Hibisci Sabdariffae, meanwhile, the present invention is isolated and purified to Calyx Hibisci Sabdariffae crude polysaccharides further by ion-exchange chromatography method, obtains four kinds of refined polysaccharide.Described crude polysaccharides and refined polysaccharide have significant immune-enhancing activity.
Description
Technical field
The invention belongs to the technical field of Chinese medicine and in particular to a kind of Calyx Hibisci Sabdariffae polysaccharide and preparation method thereof with should
With.
Background technology
Immunoregulation effect is the most important bioactive functions of polysaccharide, always the focus of people's research.Immune antibacterial
Polysaccharide and the compound of synthetic, its untoward reaction and side effect have caused people extensively to note.And most of higher plant
The polysaccharide in source is the material having no adverse reaction, and will not produce larger side effect to body, therefore, detached from plant
Polysaccharide causes great concern in biomedicine.Over one hundred kind of vegetable polysaccharidess have been carried out with the research report of active correlation at present.
It is dynamic that research shows that vegetable polysaccharidess can be regulated and controled by the signal paths different from multiple receptor bindings on immunocyte surface, activation
Immune system in thing body, including：Stimulating expression of macrophage, T/B lymphocyte, the secretion of natural killer cell or propagation；Adjust
The release of ganglion cell's factor；Promote the secretion of antibody；Activating complement system etc..
The mechanism of action of these active substances is also evolving, wherein the immunologic mechanism to non-specific induction for the polysaccharide
More it is subject to people's attention.Vegetable polysaccharidess are optimal vaccine candidate medicines.
Calyx Hibisci Sabdariffae (Hibiscus Sabdariffa L., Roselle) is that Malvaceae Hibiscuses are annual or perennial grass
This plant, also known as Hibiscus Sabdariffa Linn, very popular actor or actress certain herbaceous plants with big flowers, roselle, red sorrel etc..Roselle flower has time-honored food, medicine dual use valency
Value, the calyx of plant, seed, stem and leaf are all available.Recent study and clinical confirmation, Calyx Hibisci Sabdariffae has antioxidation, resists
Multiple medical actives such as tumor and enhancing immunologic function, lowering blood-fat and reducing weight, the liver protecting and cardiovascular system.At present, right both at home and abroad
The research of Calyx Hibisci Sabdariffae is concentrated mainly on Calyx Hibisci Sabdariffae food coloring and the activity of alcohol extraction water extract aspect, such as pigment composition is reflected
Calmly, the research such as pigment stability, water alcohol and its anthocyanin extract thing activity is more, and the research to Calyx Hibisci Sabdariffae polysaccharose substance
Rarely seen report.
Content of the invention
In order to overcome the deficiencies in the prior art and shortcoming, the primary and foremost purpose of the present invention is to provide a kind of Calyx Hibisci Sabdariffae many
The preparation method of sugar.
Another object of the present invention is to providing the Calyx Hibisci Sabdariffae polysaccharide that above-mentioned preparation method obtains.
It is still another object of the present invention to provide the application of above-mentioned Calyx Hibisci Sabdariffae polysaccharide.
The purpose of the present invention is realized by following proposal：
A kind of preparation method of Calyx Hibisci Sabdariffae crude polysaccharides, comprises the steps of：
(1) by Calyx Hibisci Sabdariffae (Hibiscus Sabdariffa L.) calyx grinding and sieving, obtain Calyx Hibisci Sabdariffae powder；Steam
Then extracting solution is centrifuged, takes supernatant liquid by distilled water heating and refluxing extraction Calyx Hibisci Sabdariffae powder, filters, and filtrate decompression is dense
Contracting, obtains polysaccharide solution；
(2) the macroporous adsorbent resin mixing by polysaccharide solution with through pretreatment, desolventing technology, are then filtered off polysaccharide molten
Liquid, and the polysaccharide of absorption in resin is repeatedly washed out with water, the polysaccharide solution after decolouring is merged, concentrating under reduced pressure, obtain concentrating sugar
Liquid；
(3) add precipitant to be precipitated in concentrating sugar liquid, be then centrifuged for, abandoning supernatant, collect precipitate and do
Dry, obtain Calyx Hibisci Sabdariffae crude polysaccharides (HSP-C).
Precipitant described in step (3) is dehydrated alcohol or ethanol solution that volume fraction is 90～100%, described precipitation
The addition of agent is concentrate sugar liquid volume 3～5 times.
The ratio of water to material that hot water return described in step (1) extracts is 15:1～30:1(m/m)；
The temperature being heated to reflux described in step (1) is 75～100 DEG C；The time that described hot water return extracts is 2～
4h, the number of times that hot water return extracts is 2～4 times；
The rotating speed of the centrifugation described in step (1) is 3000～5000r/min；The time of described centrifugation be 8～
12min；The number of times filtering is 2～4 times；
The condition of the described desolventing technology of step (2) is in 40～60 DEG C of water-bath 2～4h.
Macroporous adsorbent resin described in step (2) is selected as D354FD resin；
The method of the pretreatment of macroporous adsorbent resin described in step (2) is：Macroporous adsorbent resin is first used distilled water
Soak 12～24h, then first soak 0.5～3h with the hydrochloric acid solution that mass fraction is 3～5%, distillation is washed to neutrality, then uses
Mass fraction is 3～5% sodium hydroxide solution immersion 0.5～3h, and distillation is washed to neutrality, obtains the macropore through pretreatment
Adsorbent resin；
The temperature of concentrating under reduced pressure described in step (1) and (2) is 40～60 DEG C；
The temperature of the precipitation described in step (3) is 0～4 DEG C, and the time of precipitation is 8～24h；
The rotating speed of the centrifugation described in step (3) is 3000～5000r/min；The time of described centrifugation be 12～
15min；
The temperature of the drying described in step (3) is 40～60 DEG C, is dried to constant weight.
A kind of Calyx Hibisci Sabdariffae crude polysaccharides (HSP-C), are prepared by above-mentioned preparation method；
Four kinds of Calyx Hibisci Sabdariffae refined polysaccharide (HSP-I, HSP-II, HSP-III and HSP-IV), by by above-mentioned Calyx Hibisci Sabdariffae
Calyx crude polysaccharides are further purified and obtain.
Four kinds of described Calyx Hibisci Sabdariffae refined polysaccharide (HSP-I, HSP-II, HSP-III and HSP-IV) are all homogeneous group
Point, its molecular weight (Mn) respectively is 8733Da, 14190Da, 44408Da and 70422Da.
Described Calyx Hibisci Sabdariffae refined polysaccharide (HSP-I, HSP-II, HSP-III and HSP-IV)) make as follows
Standby obtain：
(1) the DEAE-52 resin through pretreatment is proceeded to Z-type chromatographic column after 40～60 DEG C of rotary evaporation bubble removings
In, pump distilled water with constant flow pump so that filler dress post is uniform, after balance, flow velocity 5～10s/ drips；
(2) Roselle flower crude polysaccharides are configured to polysaccharide solution, proceed in chromatographic column, use distilled water, 0.1mol/L successively
NaCl, 0.2mol/L NaCl, 0.3mol/L NaCl solution eluting, flow velocity 5～10s/ drips, and collects each stream part, phenol-sulfur respectively
Acid system tracing detection eluent, measures the absorbance under 490nm, draws elution curve, and the eluent in same absworption peak merges；
Then concentrated by rotary evaporation, dialysis, vacuum lyophilization, obtain four components after DEAE-52 ion-exchange chromatography, order respectively
Entitled HSP-I, HSP-II, HSP-III and HSP-IV；
The method of the DEAE-52 resin pretreatment described in step (1) is：By DEAE-52 resin with 4～30 DEG C of distillation
Water soaks 12～24h；Then soak 0.5～2h with the hydrochloric acid solution of 0.3～0.5mol/L, distillation is washed to neutrality；Again with 0.3
The NaOH solution of～0.5mol/L soaks 0.5～2h, and distillation is washed to neutrality, obtains the DEAE-52 resin through pretreatment；
The temperature of the concentrated by rotary evaporation described in step (2) is 40～60 DEG C.
Application in preparing medicament for immunity enhancement for the described Calyx Hibisci Sabdariffae crude polysaccharides or Calyx Hibisci Sabdariffae refined polysaccharide；
Described Calyx Hibisci Sabdariffae crude polysaccharides or Calyx Hibisci Sabdariffae refined polysaccharide can be used as new medicament for immunity enhancement, should
For treating the medicine of immuno-compromised.
A kind of medicament for immunity enhancement, containing above-mentioned Calyx Hibisci Sabdariffae crude polysaccharides or Calyx Hibisci Sabdariffae refined polysaccharide.
The principle of the present invention：The polysaccharide of plant origin by promoting mouse spleen cell proliferation and can adjust exempting from of macrophage
Epidemic disease functive reveals various beneficial pharmacological actions.Present invention discover that and confirmation vegetable polysaccharidess HSP-C, HSP-I, HSP-II,
HSP-III and HSP-IV (especially HSP-II), has significant immune-enhancing activity, and using dosage is low, in same dosage
Under toxic and side effects relatively low.
The present invention has such advantages as with respect to prior art and effect：
(1) present invention firstly discovers that Calyx Hibisci Sabdariffae crude polysaccharides and its refined polysaccharide can remarkably promote mouse boosting cell increasing
Grow, strengthen the immunoloregulation function of lymphocyte, and then improve the immune level of body；
(2) present invention firstly discovers that Calyx Hibisci Sabdariffae crude polysaccharides and refined polysaccharide can significantly increase RAW264.7 macrophage
The release of NO；
(3) present invention firstly discovers that Calyx Hibisci Sabdariffae refined polysaccharide (HSP-II) can significantly increase RAW264.7 macrophage IL-6
And the release of TNF-α；
(4) present invention firstly discovers that Calyx Hibisci Sabdariffae refined polysaccharide (HSP-II) can dramatically increase RAW264.7 macrophage
The expression of IL-1 β, iNOS and IL-6mRNA；
(5) present invention firstly discovers that Calyx Hibisci Sabdariffae refined polysaccharide (HSP-II) can dramatically increase RAW264.7 macrophage
The expression of p-ERK, p-JNK, p-P38 and p-P65, points out it may play by activating MAPKs and NF- κ Bp65 signal path
Immunoregulation effect；
(6) the dosage ratio of Calyx Hibisci Sabdariffae crude polysaccharides and refined polysaccharide is relatively low, and in effective dosage scope
Endotoxic relatively low.
Brief description
Fig. 1 is the ion-exchange chromatography elution curve of embodiment 4；
Fig. 2 is Calyx Hibisci Sabdariffae refined polysaccharide (HSP-I, HSP-II, HSP-III and the HSP- that embodiment 4 prepares
IV ultraviolet scanning spectrum figure)；
Fig. 3 is Calyx Hibisci Sabdariffae refined polysaccharide (HSP-I, HSP-II, HSP-III and the HSP- that embodiment 4 prepares
IV infrared spectrogram)；Wherein A:HSP-I；B:HAP-II；C:HAP-III；D:HAP-IV；
Fig. 4 is Calyx Hibisci Sabdariffae refined polysaccharide (HSP-I, HSP-II, HSP-III and the HSP- that embodiment 4 prepares
IV gpc chromatogram)；Wherein A:HSP-I；B:HAP-II；C:HAP-III；D:HAP-IV；
Fig. 5 is that Calyx Hibisci Sabdariffae polysaccharide (HSP-C, HSP-I, HSP-II, HSP-III and HSP-IV) increases to mouse boosting cell
The impact figure grown；Wherein figure A stimulates the impact of mouse spleen cell proliferation for Calyx Hibisci Sabdariffae refined polysaccharide, and figure B is Roselle flower
Calyx refined polysaccharide works in coordination with ConA stimulates the impact of mouse spleen cell proliferation, and figure C works in coordination with LPS for Calyx Hibisci Sabdariffae refined polysaccharide to be stimulated
The impact of mouse spleen cell proliferation；
Fig. 6 is that Calyx Hibisci Sabdariffae polysaccharide (HSP-C, HSP-I, HSP-II, HSP-III and HSP-IV) is huge to RAW264.7 to be bitten
Cell discharges the impact figure of NO；
Fig. 7 is that Calyx Hibisci Sabdariffae refined polysaccharide (HSP-II) discharges the impact figure of IL-6 to RAW264.7 macrophage；
Fig. 8 is that Calyx Hibisci Sabdariffae refined polysaccharide (HSP-II) discharges the impact figure of TNF-α to RAW264.7 macrophage；
Fig. 9 is the impact that Calyx Hibisci Sabdariffae refined polysaccharide (HSP-II) is expressed to RAW264.7 macrophage iNOS mRNA
Figure, wherein, *：Compare with control group, P<0.05；**：Compare with control group, P<0.01；
Figure 10 is the impact that Calyx Hibisci Sabdariffae refined polysaccharide (HSP-II) is expressed to RAW264.7 macrophage IL-6mRNA
Figure, wherein, *：Compare with control group, P<0.05；**：Compare with control group, P<0.01；
Figure 11 is the impact that Calyx Hibisci Sabdariffae refined polysaccharide (HSP-II) is expressed to RAW264.7 macrophage IL-1 β mRNA
Figure, wherein, *：Compare with control group, P<0.05；**：Compare with control group, P<0.01；
Figure 12 is the impact to RAW264.7 macrophage p-ERK protein expression for the Calyx Hibisci Sabdariffae refined polysaccharide (HSP-II)
Figure, wherein, *：Compare with control group, P<0.05；**：Compare with control group, P<0.01；Wherein figure A-1 is p-ERK egg
White expression exposure diagram, figure A-2 is p-ERK albumen relative expression quantity；
Figure 13 is the impact to RAW264.7 macrophage p-JNK protein expression for the Calyx Hibisci Sabdariffae refined polysaccharide (HSP-II)
Figure, wherein, *：Compare with control group, P<0.05；**：Compare with control group, P<0.01；Wherein figure B-1 is p-JNK egg
White expression exposure diagram, figure B-2 is p-JNK albumen relative expression quantity；
Figure 14 is that Calyx Hibisci Sabdariffae refined polysaccharide (HSP-II) is expressed to RAW264.7 macrophage p-P38 protein mRNA
Impact figure, wherein, *：Compare with control group, P<0.05；**：Compare with control group, P<0.01；Wherein figure C-1 is p-
P38 protein expression exposure diagram, figure C-2 is p-P38 albumen relative expression quantity；
Figure 15 is that Calyx Hibisci Sabdariffae refined polysaccharide (HSP-II) is expressed to RAW264.7 macrophage p-P65 protein mRNA
Impact figure, wherein, *：Compare with control group, P<0.05；**：Compare with control group, P<0.01；Wherein figure D-1 is p-
P65 protein expression exposure diagram, figure D-2 is p-P65 albumen relative expression quantity.
Specific embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention do not limit
In this.In embodiment, mouse macrophage RAW 264.7 is purchased from Shanghai life science institute of Chinese Academy of Sciences cell resource center；
Calyx Hibisci Sabdariffae is purchased from the peaceful medicinal material market in Guangzhou, and the place of production is Yunnan.
Embodiment 1
Calyx Hibisci Sabdariffae crude polysaccharides (HSP-C) are prepared from Calyx Hibisci Sabdariffae：
(1) cross 20 mesh sieves after Calyx Hibisci Sabdariffae is pulverized, weigh 100g, use distilled water heating and refluxing extraction, wherein, ratio of water to material
For 30:1 (mass ratio), reflux, extract, temperature is 75 DEG C, and reflux extracting time is 4h, reflux, extract, number of times 3 times；It is then combined with carrying
Take liquid, be centrifuged 10min under 4000rpm, take supernatant liquid, filter paper filtering 3 times, filtrate is subtracted with Rotary Evaporators at 40 DEG C
Pressure is concentrated into 200mL, obtains polysaccharide solution；
(2) D354FD resin is first used distilled water immersion 12h, then soaked with the hydrochloric acid solution that mass fraction is 5%
0.5h, is washed to neutrality, then the sodium hydroxide solution being 5% with mass fraction soaks 0.5h, and then distillation is washed to neutrality, obtains
Standby to the D354FD resin through pretreatment；
(3) polysaccharide solution preparing step (1) and the D354FD resin equal-volume mixing through pretreatment, are placed in
In 50 DEG C of thermostat water baths, water bath with thermostatic control 3h is decoloured, interval stirring, then goes out polysaccharide solution with the filter-cloth filtering of 200 mesh,
And the polysaccharide washing out absorption in resin is repeatedly cleaned 4 times with water, the polysaccharide liquid after decolouring is merged, with Rotary Evaporators at 40 DEG C
Under be evaporated to 200mL, obtain concentrate sugar liquid；
(4) 4 times of volume dehydrated alcohol of addition in the concentration sugar liquid that step (3) prepares, side edged magnetic agitation, in
Precipitate with ethanol 8h under the conditions of 4 DEG C, then centrifugation 15min under 4000rpm, abandoning supernatant, take out precipitate and dry in 45 DEG C, obtain
To Calyx Hibisci Sabdariffae crude polysaccharides (HSP-C).
Embodiment 2
Calyx Hibisci Sabdariffae crude polysaccharides (HSP-C) are prepared from Calyx Hibisci Sabdariffae：
(1) cross 20 mesh sieves after Calyx Hibisci Sabdariffae is pulverized, weigh 100g, then distilled water heating and refluxing extraction, wherein, ratio of water to material
30:1 (mass ratio), reflux, extract, temperature is 85 DEG C, and reflux extracting time is 3h, reflux, extract, number of times 2 times；It is then combined with extracting
Liquid, is centrifuged 8min under 3000rpm, takes supernatant liquid, filter paper filtering 2 times, filtrate is reduced pressure with Rotary Evaporators at 50 DEG C
It is concentrated into 200mL, obtain polysaccharide solution；
(2) D354FD resin is first used distilled water immersion 24h, then soaks 2h with the hydrochloric acid solution that mass fraction is 5%,
Distillation is washed to neutrality, then the sodium hydroxide solution being 5% with mass fraction soaks 2h, and distillation is washed to neutrality, obtain through
The D354FD resin of pretreatment is standby；
(3) polysaccharide solution preparing step (1) and the D354FD resin equal-volume mixing through pretreatment, are placed in
In 40 DEG C of thermostat water baths, water bath with thermostatic control 2h is decoloured, interval stirring, then goes out polysaccharide solution with the filter-cloth filtering of 200 mesh,
And repeatedly washing out, with water, the polysaccharide adsorbing in resin, the polysaccharide liquid after decolouring merges, and is reduced pressure at 50 DEG C with Rotary Evaporators
It is concentrated into 200mL, obtain concentrating sugar liquid；
(4) add the dehydrated alcohol of 3 times of volumes in the concentration sugar liquid preparing in step (3), side edged magnetic force stirs
Mix, precipitate with ethanol 12h under the conditions of 0 DEG C, then centrifugation 12min under 3000rpm, abandoning supernatant, take out precipitate in 50 DEG C
Dry, obtain Calyx Hibisci Sabdariffae crude polysaccharides (HSP-C).
Embodiment 3
Calyx Hibisci Sabdariffae crude polysaccharides (HSP-C) are prepared from Calyx Hibisci Sabdariffae：
(1) cross 20 mesh sieves after Calyx Hibisci Sabdariffae is pulverized, weigh 100g, use distilled water heating and refluxing extraction, wherein, ratio of water to material is
30:1 (mass ratio), reflux, extract, temperature is 95 DEG C, and reflux extracting time is 2h, reflux, extract, number of times 4 times, is then combined with extracting
Liquid, is centrifuged 12min under 5000r/min, takes supernatant liquid, filter paper filtering 4 times, filtrate is subtracted with Rotary Evaporators at 60 DEG C
Pressure is concentrated into 200mL, obtains polysaccharide solution；
(2) D354FD resin is first used distilled water immersion 18h, then soaks 3h with the hydrochloric acid solution that mass fraction is 5%,
Distillation is washed to neutrality, then the sodium hydroxide solution being 5% with mass fraction soaks 3h, and distillation is washed to neutrality, obtain through
The D354FD resin of pretreatment is standby；
(3) the D354FD resin equal-volume mixing by the polysaccharide solution preparing in (1) with through pretreatment, is placed in 60
In DEG C thermostat water bath, water bath with thermostatic control 4h is decoloured, interval stirring, goes out polysaccharide solution with the filter-cloth filtering of 200 mesh afterwards, and
Repeatedly wash out the polysaccharide of absorption in resin with water, the polysaccharide liquid after decolouring is merged, is reduced pressure at 60 DEG C with Rotary Evaporators dense
It is reduced to 200mL, obtain concentrating sugar liquid；
(4) in the concentration sugar liquid that step (3) prepares add 5 times of volumes dehydrated alcohol, side edged magnetic agitation,
Precipitate with ethanol 24h under the conditions of 4 DEG C, then centrifugation 14min under 5000rpm, abandoning supernatant, take out precipitate and dry in 60 DEG C
Dry, obtain Calyx Hibisci Sabdariffae crude polysaccharides (HSP-C).
Embodiment 4
The preparation of Calyx Hibisci Sabdariffae refined polysaccharide (HSP-I, HSP-II, HSP-III and HSP-IV)：
(1) DEAE-52 pretreatment：Take the DEAE-52 of 70g, with 4 DEG C of immersion 24h of distilled water, then carefully upper water is fallen
Go out, remove impurity, then soak 0.5h with the hydrochloric acid solution of 0.5mol/L, upper strata acid solution is carefully poured out, sucking filtration is to after do with steaming
Distilled water is eluted to neutrality, then soaks 0.5h with the NaOH solution of 0.5mol/L, carefully pours out upper strata alkali liquor, and sucking filtration is to after do with steaming
Distilled water is washed till neutral standby；
(2) by through pretreatment DEAE-52 after 48 DEG C of rotary evaporation bubble removings Glass rod drain proceed to 2.6 × 30cm
In the Z-type chromatographic column of specification, pump distilled water with constant flow pump so that filler dress post is uniform, after balance, 7s/ drips；
(3) weigh the Calyx Hibisci Sabdariffae crude polysaccharides (HSP-C) preparing in 100mg embodiment 1, be configured to polysaccharide molten
Liquid, Glass rod is drained in chromatographic column, uses distilled water, 0.1mol/L NaCl, 0.2mol/L NaCl, 0.3mol/L successively
NaCl solution eluting, collects each stream part automatically, and every pipe is collected about 5mL, phend-sulphuric acid tracing detection eluent, measured
Absorbance at 490nm, draws elution curve, and the eluent in same absworption peak merges；Then 40 DEG C of concentrated by rotary evaporations of eluent,
Vacuum lyophilization, wherein, HSP-c obtains four components after DEAE-52 ion-exchange chromatography, is respectively designated as HSP-
I, HSP-II, HSP-III and HSP-IV, wherein, Fig. 1 is the ion-exchange chromatography elution curve of the present embodiment.
Embodiment 5
The preparation of Calyx Hibisci Sabdariffae refined polysaccharide (HSP-I, HSP-II, HSP-III and HSP-IV)：
(1) DEAE-52 pretreatment：Take the DEAE-52 of 70g, with 20 DEG C of immersion 12h of distilled water, then carefully by upper water
Pour out, remove impurity, then soak 1h with the hydrochloric acid solution of 0.5mol/L, upper strata acid solution is carefully poured out, sucking filtration is to after do with steaming
Distilled water is eluted to neutrality, then soaks 1h with the NaOH solution of 0.5mol/L, carefully pours out upper strata alkali liquor, and sucking filtration is to after do with distillation
It is washed to neutral standby；
(2) by through pretreatment DEAE-52 after 48 DEG C of rotary evaporation bubble removings Glass rod drain proceed to 2.6 × 30cm
In the Z-type chromatographic column of specification, pump distilled water with constant flow pump so that filler dress post is uniform, after balance, 7s/ drips；
(3) weigh the Calyx Hibisci Sabdariffae crude polysaccharides (HSP-C) preparing in 100mg embodiment 1, be configured to polysaccharide molten
Liquid, Glass rod is drained in chromatographic column, uses distilled water, 0.1mol/L NaCl, 0.2mol/L NaCl, 0.3mol/L successively
NaCl solution eluting, collects each stream part automatically, and every pipe is collected about 5mL, phend-sulphuric acid tracing detection eluent, measured
Absorbance at 490nm, draws elution curve, and the eluent in same absworption peak merges；Then 50 DEG C of concentrated by rotary evaporations of eluent,
Vacuum lyophilization, wherein, HSP-c obtains four components after DEAE-52 ion-exchange chromatography, is respectively designated as HSP-
I, HSP-II, HSP-III and HSP-IV, wherein, the ion-exchange chromatography eluting result of the present embodiment is with embodiment 4.
Embodiment 6
The preparation of Calyx Hibisci Sabdariffae refined polysaccharide (HSP-I, HSP-II, HSP-III and HSP-IV)：
(1) DEAE-52 pretreatment：Take the DEAE-52 of 70g, with 25 DEG C of immersion 20h of distilled water, then carefully by upper water
Pour out, remove impurity, then soak 0.8h with the hydrochloric acid solution of 0.5mol/L, upper strata acid solution is carefully poured out, sucking filtration is used to after do
Distilled water is eluted to neutrality, then soaks 0.8h with the NaOH solution of 0.5mol/L, carefully pours out upper strata alkali liquor, and sucking filtration is used to after do
Distillation is washed to neutral standby；
(2) by through pretreatment DEAE-52 after 48 DEG C of rotary evaporation bubble removings Glass rod drain proceed to 2.6 × 30cm
In the Z-type chromatographic column of specification, pump distilled water with constant flow pump so that filler dress post is uniform, after balance, 7s/ drips；
(3) weigh the Calyx Hibisci Sabdariffae crude polysaccharides (HSP-C) preparing in 100mg embodiment 1, be configured to polysaccharide solution, glass
Glass rod is drained in chromatographic column, uses distilled water, 0.1mol/L NaCl, 0.2mol/L NaCl, 0.3mol/L NaCl solution successively
Eluting, collects each stream part automatically, and every pipe is collected about 5mL, phend-sulphuric acid tracing detection eluent, measured at 490nm
Absorbance, the eluent drawn in the same absworption peak of elution curve merges；Then 60 DEG C of concentrated by rotary evaporations of eluent, vacuum freezing is done
Dry, wherein, HSP-c obtains four components after DEAE-52 ion-exchange chromatography, is respectively designated as HSP-I, HSP-II,
HSP-III and HSP-IV, wherein, the ion-exchange chromatography eluting result of the present embodiment is with embodiment 4.
Embodiment 7 UV scanning collection of illustrative plates and IR spectrum scanning
(1) prepare in Example 4～6 respectively Calyx Hibisci Sabdariffae refined polysaccharide (HSP-I, HSP-II, HSP-III and
HSP-IV) 4mg, is configured to the polysaccharide solution of 1mg/mL, with distilled water for comparison, surveys its absorbance at 190～500nm.
(2) prepare in Example 4～6 respectively Calyx Hibisci Sabdariffae refined polysaccharide (HSP-I, HSP-II, HSP-III and
HSP-IV) 2mg, mixes finely ground uniformly lamelliform, in 4000～400cm with KBr-1Carry out infrared scan in wave-number range.
Fig. 2 is Calyx Hibisci Sabdariffae refined polysaccharide (HSP-I, HSP-II, HSP-III and the HSP- that embodiment 4 prepares
IV ultraviolet scanning spectrum figure)；Fig. 3 be embodiment 4 prepare Calyx Hibisci Sabdariffae refined polysaccharide (HSP-I, HSP-II,
HSP-III and HSP-IV) infrared spectrogram；Wherein A:HSP-I；B:HAP-II；C:HAP-III；D:HAP-IV.
Interpretation of result：
(1) polysaccharide ultraviolet spectral analysis result：The Calyx Hibisci Sabdariffae refined polysaccharide preparing in embodiment 4 all 200～
280nm has stronger absorption, shows may contain unsaturated carbonyl and carboxyl in sample, all no substantially inhales in 280nm and 260nm
Receive peak, four kinds of polysaccharide are described all without protein and nucleic acid, or both contents all fewer (Fig. 2).Embodiment 5 and embodiment
6 testing result is with embodiment 4.
(2) polysaccharide results of IR：
The Calyx Hibisci Sabdariffae refined polysaccharide HSP-I (Fig. 3-A) preparing in embodiment 4, HSP-II (Fig. 3-B), HSP-III
(Fig. 3-C) and HSP-IV (Fig. 3-D) successively wave number 3399,3393,3398,3398cm-1The strong absworption peak at place is carbohydrate molecule
Between or intramolecular O-H stretching vibration peak, wave number 2927,2930,2932,2932cm-1Place exist slightly weak absworption peak be
The stretching vibration of the C-H of methine or methyl, in wave number 1417cm-1And 1331cm-1The neighbouring bimodal change angular oscillation for C-H,
This is the characteristic absorption peak of saccharide compound.Four kinds of polysaccharide are in wave number 1647～1620cm-1Place's obvious absorption peaks are carbonyl C=O
Stretching vibration, 1200～1000cm- in addition1C-OH stretching vibration, confirm HSP-I, HSP-II, HSP-III and HSP-IV
In contain alduronic acid, further illustrating four kinds of polysaccharide is all acidic polysaccharose.Additionally, four kinds of polysaccharide are in 1200～1000cm-1Have
3～4 absworption peaks represent that HSP-I, HSP-II, HSP-III and HSP-IV sugar ring configuration is that (furan type here interval has pyranoid form
Two absworption peaks).In wave number 917cm-1And 890cm-1Neighbouring absworption peak is that the asymmetric stretching vibration of amylene oxide ring is made
The C-O-C skeletal vibration becoming and the change angular oscillation of β-D- glucopyanosyl C-H, in 764cm-1Locate as D- Glucopyranose. ring
Vibration, shows four components all containing β-D- Glucopyranose. ring.Additionally, four kinds of refined polysaccharide of Calyx Hibisci Sabdariffae are in 820cm-1
Characteristic absorption peak, represent there is rhamnoside (Fig. 3).The testing result of embodiment 5 and embodiment 6 is with embodiment 4.
Embodiment 8 HSP-I, the molecular weight of HSP-II, HSP-III and HSP-IV and Purity
Respectively embodiment 4～6 is prepared using high performance liquid chromatograph Calyx Hibisci Sabdariffae refined polysaccharide (HSP-I,
HSP-II, HSP-III and HSP-IV) 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 the KH of 0.02mol/L2PO4Slow
Rush solution, flow velocity is 0.6mL/min, 2414 Composition distribution, 35 DEG C of column temperature.
Interpretation of result：
The Calyx Hibisci Sabdariffae refined polysaccharide (HSP-I, HSP-II, HSP-III and HSP-IV) that embodiment 4 prepares
Gpc chromatogram is as shown in Figure 4.The HSP-I (Fig. 4-A) that embodiment 4 prepares, HSP-II (Fig. 4-B), HSP-III (Fig. 4-C) and
The GPC figure of HSP-IV (Fig. 4-D) is single symmetrical peak, shows that HSP-I, HSP-II, HSP-III and HSP-IV are homogeneous many
Sugar, its peak position molecular mass (Mp) is respectively 8733Da, 14190Da, 44408Da and 137448Da；Equal molecular mass (the M of numbern)
It is respectively 513Da, 10431Da, 32418Da and 113870Da；Weight average molecular mass (Mw) it is respectively 8072Da, 18667Da,
50978Da and 175780Da；Z equal molecular mass (Mz) it is respectively 10984Da, 29174Da, 70422Da and 307262Da (figure
4).Embodiment 5 and 6 results are with embodiment 4.
Embodiment 9 Calyx Hibisci Sabdariffae Polysaccharides on Mice Spleen cell proliferation affects
Prepare mouse boosting cell suspension：First make mice daze with ether, so that its cervical dislocation is put to death with aseptic nipper, then
It is totally submerged after mice 2min with 75% ethanol, be transferred to and isolate mouse spleen under superclean bench, aseptic condition.To divide
From spleen be placed in above immersed with 200 purposes of aseptic PBS solution, uniformly ground with Glass rod.Afterwards with 1000rpm from
Heart 5min precipitates spleen cell, abandons supernatant, adds the lysate having configured, and bus dropper adds after blowing and beating 1～2min immediately
The PBS entering 4mL is in order to balance the osmotic pressure of spleen cell.Cell suspension is centrifuged 5min in 1000rpm again, abandons supernatant afterwards
Dissolved with appropriate RPMI-1640 complete culture solution afterwards, the piping and druming of bus dropper uniformly, that is, obtains the single spleen cell of mice and hangs
Liquid.Blood cell counting plate counts, and dilutes adjustment splenocyte concentration for 200,000/mL with RPMI-1640 complete culture solution, 50 μ L/
Hole is inoculated in 96 orifice plates, then is separately added into each concentration polysaccharide sample (each polysaccharide in terms of 100 μ L solution systems in 40 μ L/ holes
Sample concentration is 50,100,200,400 μ g/mL) (the Calyx Hibisci Sabdariffae crude polysaccharides HSP-C that embodiment 1 prepares, and implement
Example 4 prepares Calyx Hibisci Sabdariffae refined polysaccharide HSP-I, HSP-II, HSP-III and HSP-IV), 6 multiple holes of each concentration.By following
Mode is administered packet：HSP sample solution (each polysaccharide in terms of 100 μ L solution systems of complete culture solution+40 μ L in 10 μ L/ holes
Sample concentration is 50,100,200,400 μ g/mL) group；The HSP sample in LPS (final concentration 10 μ g/mL)+40 μ L/ holes in 10 μ L/ holes
Solution coordinated groups；The HSP sample solution coordinated groups in ConA (final concentration 5 μ g/mL)+40 μ L/ holes in 10 μ L/ holes；Complete with 40 μ L/ holes
Full nutrient solution replaces each concentration polysaccharide sample, is separately added into 10 μ L complete culture solutions as blank group, add 10 μ L LPS or
ConA, as comparison, separately sets 100 μ L complete culture solutions and is used for the group that returns to zero.Each concentration of each sample sets 6 multiple holes.After administration,
96 orifice plates are placed in 37 DEG C, 5%CO2Constant temperature culture 68h in cell culture incubator, the MTT work of the 5mg/mL of each hole addition 20 μ L afterwards
Make liquid.After continuing culture 4h in the incubator, add acid isopropyl alcohol by 100 μ L/ holes, place 12h, setting microplate reader program shake
Measure the absorbance in each hole under 570nm wavelength after swinging 60s.
Fig. 5 is that Calyx Hibisci Sabdariffae polysaccharide (HSP-C, HSP-I, HSP-II, HSP-III and HSP-IV) increases to mouse boosting cell
The impact figure grown；Wherein figure A stimulates the impact of mouse spleen cell proliferation for Calyx Hibisci Sabdariffae refined polysaccharide, and figure B is Roselle flower
Calyx refined polysaccharide works in coordination with ConA stimulates the impact of mouse spleen cell proliferation, and figure C works in coordination with LPS for Calyx Hibisci Sabdariffae refined polysaccharide to be stimulated
The impact of mouse spleen cell proliferation.
The crude polysaccharides HSP-C of embodiment 1 preparation, embodiment 4 prepares Calyx Hibisci Sabdariffae refined polysaccharide HSP-I, HSP-II,
HSP-III and HSP-IV can promote the mouse spleen cell proliferation of ConA and LPS induction.
Embodiment 10 Griess method detects HSP-C, and HSP-I, HSP-II, HSP-III and HSP-IV are to RAW264.7 cell
The impact of release NO
Cellar culture cell, the RAW264.7 cell of trophophase of taking the logarithm, piping and druming cell becomes 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%CO2Incubator is cultivated, adherent 24h, inhales and abandons supernatant,
By following packet requirement administration, every group sets 3 multiple holes：Control group, lipopolysaccharide (LPS, final concentration of 1 μ g/mL) group, polysaccharide
Sample sets (the Calyx Hibisci Sabdariffae crude polysaccharides HSP-C that embodiment 1 prepares, and embodiment 4 prepares Calyx Hibisci Sabdariffae refined polysaccharide
HSP-I, HSP-II, HSP-III and HSP-IV, final concentration of 31.25,62.5,125,250,400,500 μ g/mL) group, comparison
Group adds the culture medium of same volume.After administration, it is placed in 37 DEG C, 5%CO2Incubator, continues culture 24h.Take each hole cell respectively
Supernatant 100 μ L, accordingly adds in another 96 new orifice plates.Every hole adds 50 μ L Griess reagent A and 50 μ L Griess reagent B,
It is subsequently placed in reaction 10min in 37 DEG C of incubators, puts plate immediately on multiple labeling micropore board detector, examine under 550nm wavelength
Survey each hole absorbance.
Fig. 6 is that Calyx Hibisci Sabdariffae polysaccharide (HSP-C, HSP-I, HSP-II, HSP-III and HSP-IV) is huge to RAW264.7 to be bitten
Cell discharges the impact figure of NO.
HSP-C, HSP-I, HSP-II, HSP-III and HSP-IV can promote the release of RAW264.7 macrophage NO, especially
It is HSP-II most pronounced effects and assumes obvious dose dependent (Fig. 6).
Embodiment 11 Elisa method detects HSP-I, and HSP-II, HSP-III and HSP-IV discharge IL- to RAW264.7 cell
6 and the impact of TNF-α
Cellar culture cell, the RAW264.7 cell of trophophase of taking the logarithm, piping and druming cell becomes single cell suspension, under microscope
After being counted with blood cell counting plate, 1000r/min, centrifugation 5min removes supernatant, and culture medium is resuspended and adjusts cell concentration, by 200,000
The cell density in individual/hole is inoculated in 24 well culture plates, is placed in 37 DEG C, 5%CO2Incubator is cultivated, adherent 24h, inhales and abandons supernatant, presses
Following packet requires administration, and every group sets 3 multiple holes：Control group, lipopolysaccharide (LPS, final concentration of 1 μ g/mL) group, many sugar-likes
Product group (the Calyx Hibisci Sabdariffae refined polysaccharide HSP-II that embodiment 4 prepares, final concentration of 31.25,62.5,125,250,500 μ g/
), mL matched group adds the culture medium of same volume.After administration, it is placed in 37 DEG C, 5%CO2Incubator, continues culture 24h.Take respectively
Each hole cell conditioned medium detects cytokine according to Mouse IL-6ELISA Kit and Mouse TNF-α ELISA Kit operational approach
Release conditions.
Fig. 7 is that Calyx Hibisci Sabdariffae refined polysaccharide (HSP-II) discharges the impact figure of IL-6 to RAW264.7 macrophage；Fig. 8
It is that Calyx Hibisci Sabdariffae refined polysaccharide (HSP-II) discharges the impact figure of TNF-α to RAW264.7 macrophage.
HSP-II can promote RAW264.7 macrophage IL-6 (Fig. 7) and the release of TNF-α (Fig. 8), and assumes gradient
Dependency.
The impact that embodiment 12 HSP-II expresses to RAW264.7 cell iNOS, IL-6 and IL-1 β mRNA
Cellar culture cell, takes the cell of opposite long number phase, is inoculated in 6 orifice plates 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 group, lipopolysaccharide (LPS, final concentration of 1 μ
G/mL) group, HSP-II group (the Calyx Hibisci Sabdariffae refined polysaccharide that embodiment 4 prepares, final concentration of 62.5,125,250,500 μ g/
ML), matched group adds the culture medium of same volume.After administration, it is placed in 37 DEG C, 5%CO2Incubator, continues culture 24h.Effect 24h
Afterwards, inhale and abandon cell conditioned medium, PBS washes 2 times.Every hole adds Trizol 1mL, stands 5min, and suction pipe is blown and beaten to liquid no dope,
Draw cell pyrolysis liquid and proceed to EP pipe, often pipe adds 0.2mL chloroform, cover EP lid, the hand-held concussion 10s up and down that exerts oneself, room temperature
Standing 5min, 4 DEG C are centrifuged 15min with 12,000rpm, and after centrifugation, transfer upper strata aqueous phase, to another EP pipe, adds 0.5mL isopropyl
Alcohol, room temperature stands 10min, and 4 DEG C of centrifuges are centrifuged 10min with 12,000rpm, carefully inhale supernatant discarded, with cold 75% ethanol 1mL
Cleaning 2 times, is respectively centrifuged 5min with 7,500rpm under the conditions of 4 DEG C, careful suction abandons supernatant, 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 use RevertAid
First Strand cnthesis Kit (Thermo company) reverse transcription reagent box, 20 μ L reaction systems, reverse to RNA
Record.Using DyNAmo Fla sh SYRB Green qPCR Kit (Thermo company) test kit, ABI real-time fluorescence quantitative PCR
Instrument (Rrism 7500, Applied Biosystems, Foster City, CA, USA) expands to mRNA, uses after amplification
In ABI PRISM 7500SDS software, Relative Quantification (ddCt) Study method is automatically analyzed to obtain
The relative expression quantity of genes of interest.Related gene mRNA primer sequence be GAPDH ((forward, 5 '-
TTTGTCAAGCTCATTTCCTGGTATG-3′,reverse,5′-TGGGATAGGGCCTCTCTTGC-3′),IL-1β
(forward,5′-TGAAGGGCTGCTTCCAAACCTTTGACC-3′,reverse,5′-
TGTCCATTGAGGTGGAGAGCTTTCAGC-3′),IL-6(forward,5′-TACTCGGCAAACCTAGTGCG-3′,
reverse,5′–GTGTCCCAACATTCATATTGTCAGT-3′),iNOS(forward,5′-CGGCAA
ACATGACTTCAGGC-3′,reverse,5′-GCACATCAAAGCGGCCATAG-3′).
Fig. 9 is the impact that Calyx Hibisci Sabdariffae refined polysaccharide (HSP-II) is expressed to RAW264.7 macrophage iNOS mRNA
Figure, wherein, *：Compare with control group, P<0.05；**：Compare with control group, P<0.01；Figure 10 is Calyx Hibisci Sabdariffae essence
The impact figure that polysaccharide (HSP-II) processed is expressed to RAW264.7 macrophage IL-6mRNA, wherein, *：Compare with control group, P
<0.05；**：Compare with control group, P<0.01；Figure 11 is that Calyx Hibisci Sabdariffae refined polysaccharide (HSP-II) is huge to RAW264.7
The impact figure of phagocyte IL-1 β mRNA expression, wherein, *：Compare with control group, P<0.05；**：Compare with control group,
P<0.01.
Compared with control group, HSP-II can remarkably promote iNOS (Fig. 9), IL-6 (Figure 10) and IL-1 β (Figure 11)
Secretion (P ＜ 0.05), and assume gradient dependency.
The impact to RAW264.7 cell p-ERK, p-JNK, P-p38 and p-P65 protein expression for embodiment 13 HSP-II
Cellar culture cell, takes the RAW264.7 cell of opposite long number phase, is inoculated in 6 orifice plates by 1,000,000/hole cell number,
After incubation 24h, inhale and abandon supernatant, by following packet requirement administration, every group sets 3 multiple holes.Control group, and lipopolysaccharide (LPS, dense eventually
Spend for 1 μ g/mL) group, HSP-II group (the Calyx Hibisci Sabdariffae refined polysaccharide that embodiment 4 prepares, final concentration of 125,250,500 μ g/
ML), matched group adds the culture medium of same volume.After administration, it is placed in 37 DEG C, 5%CO2Incubator, continues culture 24h.After 24h,
Cell conditioned medium is abandoned in suction, and with the PBS of pre-cooling twice, collects cell to 1.5mL centrifuge tube, add the PIPA of 60 μ L to split
Solution liquid (phosphoric acid enzyme inhibitor (PhosSTOP, Roche) and protease inhibitor (cOmplete ULTRA Tablets,
Mini, EDTA-free, EASYpack, Roche)), draw lysate with the syringe of 1mL and repeatedly blow and beat cell to fully mixed
Even, crack 40min on ice, and every 10min, 15-20s is vibrated on turbula shaker.In 4 DEG C, 12000rpm is centrifuged
20min, supernatant is transferred to new 1.5mL EP pipe, then adopts BCA method detection protein concentration (according to BCA Protein
Assay Kit description is carried out)：First by concentration the BSA solution for 2mg/mL to give ultra-pure deionized water to be diluted to series mass dense
The standard solution of degree (0 μ g/mL, 25 μ g/mL, 125 μ g/mL, 250 μ g/mL, 500 μ g/mL, 1000 μ g/mL), further according to need
Configure a certain amount of working solution (BCA Solution:4% (volume fraction) Cupric Sulfate=200:4).Take one piece
96 orifice plates of no substrate, every hole is separately added into the serial standards solution 25 μ L having diluted, and need detection albumen molten
Liquid 25 μ L (having diluted 10 times), every group is respectively provided with multiple holes.It is simultaneously introduced 200 μ L/ hole working solutions, gently concussion mixes, and is placed in 37
DEG C incubation 30min after, take out, in microplate reader 570nm at detection OD value.According to standard substance protein solution surveyed OD value drafting standard
Curve, calculates testing protein solution concentration.The protein concentration being recorded according to BCA method, calculates 40 μ g total protein desirable proteins
Volume, simultaneously plus 5 μ L 5 × SDS-PAGE sample-loading buffers, ultra-pure deionized water to the cumulative volume being supplemented with respective volume is 25
μ L, mixes, and is fully centrifuged, makes liquid accumulation in bottom, 100 DEG C of metal bath heat denatured 10min, and centrifugation is placed in standby on ice.
Select 10% (volume fraction) separation gel and 5% (volume fraction) to concentrate glue, the good protein sample of degeneration is sequentially added respectively
Swimming lane, the swimming lane of sample both sides is separately added into 5 μ L Marker.The TGS buffer of q.s, deposition condition is added in electrophoresis tank
For：100V, about 150min, until bromophenol blue indicator run to away from glue lower edge about 0.5cm when, stop electrophoresis.Then in ice bath
Voltage transferring film 100min with 100V.With the TBST closing containing 5% (mass fraction) defatted milk powder, slowly sway 1h.One is anti-
Body is respectively：GAPDH (14c10) Rabbit mAb, pERK p-JNK, p-P38 and NF- Κ b p-P65 (are purchased from CST public
Department), two anti antibodys are goat antirabbit (HRP labelling), purchased from Ju Yan bio tech ltd.The dilution recommended to specifications
Ratio (1:1000), prepare one to resist, under room temperature, jog is incubated 4h or 4 DEG C and stands overnight.After one anti-incubation terminates, change two and resist,
The two of HRP labelling are anti-to press corresponding proportion dilution (1:2000), room temperature jog 1h.After two resistive connection bundles, luminescent solution is added (to be purchased from
BioFuture company) and using the development of gel imaging instrument protein band.
Figure 12 is the impact to RAW264.7 macrophage p-ERK protein expression for the Calyx Hibisci Sabdariffae refined polysaccharide (HSP-II)
Figure, wherein, *：Compare with control group, P<0.05；**：Compare with control group, P<0.01；Wherein figure A-1 is p-ERK egg
White expression exposure diagram, figure A-2 is p-ERK albumen relative expression quantity.Figure 13 is that Calyx Hibisci Sabdariffae refined polysaccharide (HSP-II) is right
The impact figure of RAW264.7 macrophage p-JNK protein expression, wherein, *：Compare with control group, P<0.05；**：With
Control group compares, P<0.01；Wherein figure B-1 is p-JNK protein expression exposure diagram, and figure B-2 is p-JNK albumen relative expression
Amount.Figure 14 is the impact that Calyx Hibisci Sabdariffae refined polysaccharide (HSP-II) is expressed to RAW264.7 macrophage p-P38 protein mRNA
Figure, wherein, *：Compare with control group, P<0.05；**：Compare with control group, P<0.01；Wherein figure C-1 is p-P38 egg
White expression exposure diagram, figure C-2 is p-P38 albumen relative expression quantity.Figure 15 is that Calyx Hibisci Sabdariffae refined polysaccharide (HSP-II) is right
The impact figure of RAW264.7 macrophage p-P65 protein mRNA expression, wherein, *：Compare with control group, P<0.05；**：With
Control group compares, P<0.01；Wherein figure D-1 is p-P65 protein expression exposure diagram, and figure D-2 is p-P65 albumen relative expression
Amount.
In normal group RAW264.7 macrophage, ERK, JNK, P38 and P65 protein expression of phosphorylation is relatively low, when difference is dense
After the HSP-II (final concentration of 125,250,500 μ g/mL) of degree and LPS (final concentration of 1 μ g/mL) function cells 24h, intracellular
The ERK (Figure 12) of phosphorylation, JNK (Figure 13), P38 (Figure 14) and P65 (Figure 15) protein expression significantly raise (P ＜ 0.01), from
And activate MAPK and NF- κ B signal path (Figure 12～15).
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not subject to above-described embodiment
Limit, other any spirit without departing from the present invention and the change made under principle, modification, replacement, combine, simplify,
All should be equivalent substitute mode, be included within protection scope of the present invention.
SEQUENCE LISTING
<110>South China Science ＆ Engineering University
<120>A kind of Calyx Hibisci Sabdariffae polysaccharide and preparation method and application
<130> 1
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223>The forward of GAPDH
<400> 1
tttgtcaagc tcatttcctg gtatg 25
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>The reverse of GAPDH
<400> 2
tgggataggg cctctcttgc 20
<210> 3
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223>The forward of IL-1 β
<400> 3
tgaagggctg cttccaaacc tttgacc 27
<210> 4
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223>The reverse of IL-1 β
<400> 4
tgtccattga ggtggagagc tttcagc 27
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>The forward of IL-6
<400> 5
tactcggcaa acctagtgcg 20
<210> 6
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223>The reverse of IL-6
<400> 6
gtgtcccaac attcatattg tcagt 25
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>The forward of iNOS
<400> 7
cggcaaacat gacttcaggc 20
<210> 8
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>The reverse of iNOS
<400> 8
gcacatcaaa gcggccatag 20
Claims (10)
1. a kind of preparation method of Calyx Hibisci Sabdariffae crude polysaccharides it is characterised in that：Comprise the steps of：
(1) by Calyx Hibisci Sabdariffae grinding and sieving, obtain Calyx Hibisci Sabdariffae powder；Distilled water heating and refluxing extraction Calyx Hibisci Sabdariffae
Then extracting solution is centrifuged by powder, takes supernatant liquid, filters, filtrate reduced in volume obtains polysaccharide solution；
(2) the macroporous adsorbent resin mixing by polysaccharide solution with through pretreatment, desolventing technology, are then filtered off polysaccharide solution,
And repeatedly washing out, with water, the polysaccharide adsorbing in resin, the polysaccharide solution after decolouring merges, concentrating under reduced pressure, obtains concentrating sugar liquid；
(3) add precipitant to be precipitated in concentrating sugar liquid, be then centrifuged for, abandoning supernatant, collect precipitate and be dried, obtain
To Calyx Hibisci Sabdariffae crude polysaccharides.
2. Calyx Hibisci Sabdariffae crude polysaccharides according to claim 1 preparation method it is characterised in that：
The temperature being heated to reflux described in step (1) is 75～100 DEG C；The time that described hot water return extracts is 2～4h；Step
Suddenly the number of times that in (1), hot water return extracts is 2～4 times；
The condition of the described desolventing technology of step (2) is in 40～60 DEG C of water-bath 2～4h；
Precipitant described in step (3) is dehydrated alcohol or ethanol solution that volume fraction is 90～100%；
The temperature of the precipitation described in step (3) is 0～4 DEG C.
3. Calyx Hibisci Sabdariffae crude polysaccharides according to claim 1 preparation method it is characterised in that：Described in step (2)
Macroporous adsorbent resin from be D354FD resin；
The method of the pretreatment of macroporous adsorbent resin described in step (2) is：Macroporous adsorbent resin is first used distilled water immersion
12～24h, then first soaks 0.5～3h with the hydrochloric acid solution that mass fraction is 3～5%, distillation is washed to neutrality, then uses quality
Fraction is 3～5% sodium hydroxide solution immersion 0.5～3h, and distillation is washed to neutrality, obtains the macroporous absorption through pretreatment
Resin.
4. Calyx Hibisci Sabdariffae crude polysaccharides according to claim 1 preparation method it is characterised in that：Described in step (3)
The addition of precipitant is concentrate sugar liquid volume 3～5 times；The time of precipitation described in step (3) is 8～24h；
The ratio of water to material that hot water return described in step (1) extracts is 15:1～30:1；
The temperature of the concentrating under reduced pressure described in step (1) and (2) is 40～60 DEG C.
5. Calyx Hibisci Sabdariffae crude polysaccharides according to claim 1 preparation method it is characterised in that：
The rotating speed of the centrifugation described in step (1) is 3000～5000r/min；The time of described centrifugation is 8～12min；Institute
The number of times stating filtration is 2～4 times；
The rotating speed of the centrifugation described in step (3) is 3000～5000r/min；The time of described centrifugation is 12～15min；Step
Suddenly the temperature of the drying described in (3) is 40～60 DEG C.
6. the Calyx Hibisci Sabdariffae crude polysaccharides that the preparation method described in a kind of any one by Claims 1 to 5 obtains.
7. four kinds of Calyx Hibisci Sabdariffae refined polysaccharide it is characterised in that：Entered by the Calyx Hibisci Sabdariffae crude polysaccharides described in claim 6
One step purification obtains；
Four kinds of described Calyx Hibisci Sabdariffae refined polysaccharide are designated as HSP-I, HSP-II, HSP-III and HSP-IV respectively, four kinds of Flos Rosae Rugosaes
Flos Solani Melongenae calyx refined polysaccharide is all homogeneous components, and its Mn molecular weight is respectively：HSP-I molecular weight is 8733Da, HSP-II molecular weight
For 14190Da, HSP-III molecular weight is 44408Da, and HSP-IV molecular weight is 70422Da.
8. four kinds of Calyx Hibisci Sabdariffae refined polysaccharide according to claim 7 it is characterised in that：It is made by the steps
Arrive：
(1) the DEAE-52 resin through pretreatment is proceeded in Z-type chromatographic column after 40～60 DEG C of rotary evaporation bubble removings, use
So that filler dress post is uniform, after balance, flow velocity 5～10s/ drips constant flow pump pumping distilled water；
(2) Roselle flower crude polysaccharides are configured to polysaccharide solution, proceed in chromatographic column, use distilled water, 0.1mol/L successively
NaCl, 0.2mol/L NaCl, 0.3mol/L NaCl solution eluting, flow velocity 5～10s/ drips, and collects each stream part, phenol-sulfur respectively
Acid system tracing detection eluent, measures the absorbance under 490nm, draws elution curve, and the eluent in same absworption peak merges；
Then concentrated by rotary evaporation, dialysis, vacuum lyophilization, obtain four components after DEAE-52 ion-exchange chromatography, remember respectively
For HSP-I, HSP-II, HSP-III and HSP-IV.
9. four kinds of Calyx Hibisci Sabdariffae refined polysaccharide according to claim 8 it is characterised in that：
The method of the DEAE-52 resin pretreatment described in step (1) is：By DEAE-52 resin with 4～30 DEG C of distillation water logging
Bubble 12～24h；Then soak 0.5～2h with the hydrochloric acid solution of 0.3～0.5mol/L, distillation is washed to neutrality；Again with 0.3～
The NaOH solution of 0.5mol/L soaks 0.5～2h, and distillation is washed to neutrality, obtains the DEAE-52 resin through pretreatment；
The temperature of the concentrated by rotary evaporation described in step (2) is 40～60 DEG C.
10. Calyx Hibisci Sabdariffae crude polysaccharides according to claim 6 or the Roselle flower described in any one of claim 7～9
Application in preparing medicament for immunity enhancement for the calyx refined polysaccharide.
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