CN106519055B

CN106519055B - A kind of calyx polysaccharide and the preparation method and application thereof from generation to generation

Info

Publication number: CN106519055B
Application number: CN201611001914.5A
Authority: CN
Inventors: 姜建国; 申春燕; 宋德幸
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2016-11-11
Filing date: 2016-11-11
Publication date: 2019-08-20
Anticipated expiration: 2036-11-11
Also published as: CN106519055A

Abstract

The invention belongs to the field of Chinese medicines, disclose a kind of calyx polysaccharide and the preparation method and application thereof from generation to generation.The present invention extracted by hot water return, decolorization and alcohol precipitation, and calyx Thick many candies from generation to generation are extracted from calyx from generation to generation.By that calyx Thick many candies will be further purified from generation to generation, refined polysaccharide is obtained.Thick many candies and refined polysaccharide of the invention have anti-oxidant, antitumor and immune-enhancing activity, and dosage is low, and the toxic side effect under same dosage is relatively low.

Description

A kind of calyx polysaccharide and the preparation method and application thereof from generation to generation

Technical field

The invention belongs to the technical fields of Chinese medicine, and in particular to a kind of calyx polysaccharide and the preparation method and application thereof from generation to generation.

Background technique

Polysaccharide is the important biological polymeric compound of one kind other than protein and nucleic acid.Modern pharmacological research table Bright polysaccharide has a variety of physiological activity, including anti-oxidant, antitumor, antiviral, reducing blood lipid, anti-aging, enhancing immune function Deng.Immunoregulation effect is the most important bioactive functions of polysaccharide, always is the hot spot of research.Immune bacterial polysaccharides and Artificial synthesized compound, adverse reaction and side effect have caused people to pay attention to extensively.And most of higher plant sources Polysaccharide is the substance having no adverse reaction, and will not generate biggish side effect to body, therefore, the polysaccharide separated from plant exists It causes great concern in biomedicine.Active relevant research is carried out to over one hundred kind of plant polyose at present to report.Study table Bright plant polyose can by conjunction with a variety of receptors on immunocyte surface, signal path that activation is different regulate and control animal body Interior immune system, comprising: the secretion or proliferation of stimulating expression of macrophage, T/B lymphocyte, natural killer cells；Adjust cell The release of the factor；Promote the secretion of antibody；Activating complement system etc..

The mechanism of action of these active materials is also evolving, wherein immunologic mechanism of the polysaccharide to non-specificity induction More it is valued by people.Plant polyose is optimal vaccine candidate drug.

(Citrus aurantium L.var.amara Engl) is a change of Rutaceae citrus plant bitter orange from generation to generation Kind, bitter orange flower is the dry product of its bud, has good pharmacological action, is usually used in treating depression of QI, abdominal distention, having indigestion not Change, nausea and vomiting etc..At present both at home and abroad to the research of bitter orange flower mainly based on its volatile oil compound, mainly steamed using water Steam distillation method refines essential oil, and is widely used in the industries such as cosmetics, essence, fragrance.And to the research report of bitter orange flower polysaccharide Road almost without.Most of natural activity polysaccharide are the natural green products having no toxic side effect.With the continuous deepening of research, day Right polysaccharide increasingly become food, health care product, medicine and other fields research hotspot.

Summary of the invention

For overcome the deficiencies in the prior art and disadvantage, the primary purpose of the present invention is that provide a kind of calyx from generation to generation slightly more The preparation method of sugar and refined polysaccharide.

The Thick many candies of calyx from generation to generation and purification being prepared another object of the present invention is to provide above-mentioned preparation method are more Sugar.

A further object of the present invention is to provide the applications of the above-mentioned Thick many candies of calyx from generation to generation and refined polysaccharide.

The purpose of the present invention is realized by following proposal:

A kind of preparation method of the Thick many candies of calyx from generation to generation, comprises the following steps:

(1) will (Citrus aurantium L.var.amara Engl) calyx grinding and sieving from generation to generation, obtain bitter orange flower Calyx coarse powder；Extracting solution, is then centrifuged, and supernatant is concentrated under reduced pressure, obtains by distilled water heating and refluxing extraction calyx coarse powder from generation to generation To the Thick many candies solution of calyx from generation to generation of concentration；

(2) it being mixed by polysaccharide solution and by pretreated macroporous absorbent resin, decolorization filters out polysaccharide solution, And wash out the polysaccharide adsorbed in resin repeatedly with water, the polysaccharide solution after decoloration is merged, is concentrated under reduced pressure, concentration liquid glucose is obtained；

(3) precipitating reagent is added in concentration liquid glucose to be precipitated, is then centrifuged for, discard supernatant liquid, collect sediment and do It is dry, obtain calyx Thick many candies (being denoted as CAVAPs) from generation to generation.

The number that hot water return extracts in step (1) is 2~4 times；The temperature being heated to reflux described in step (1) be 75~ 100℃；The time that the hot water return extracts every time is 2~4h；Hot water return described in step (1) extract ratio of water to material be 15:1~30:1 (mass ratio).

The temperature of reduced pressure described in step (1) and (2) is 40~60 DEG C.

The revolving speed of centrifugation described in step (1) is 3000~5000r/min；The time of the centrifugation is 8~12min； The number of the filtering is 2~4 times.

The condition of step (2) described decolorization is in 40~60 DEG C of 2~4h of water-bath；

It is D354FD resin that macroporous absorbent resin described in step (2), which is selected,；

The pretreated method of macroporous absorbent resin described in step (2) are as follows: by macroporous absorbent resin first with distillation water logging Bubble 12~for 24 hours, 0.5~3h first then is impregnated with the hydrochloric acid solution that mass fraction is 3%~5%, distillation is washed to neutrality, then uses The sodium hydroxide solution that mass fraction is 3%~5% impregnates 0.5~3h, and distillation is washed to neutrality, obtains by pretreated big Macroporous adsorbent resin.

The ethanol solution that precipitating reagent is dehydrated alcohol or volume fraction is 90%~100% described in step (3)；

The temperature of precipitating described in step (3) is 0~4 DEG C.

The additional amount of precipitating reagent described in step (3) is 3~5 times that liquid glucose volume is concentrated；Precipitating described in step (3) Time be 8~for 24 hours.

The revolving speed of centrifugation described in step (3) is 3000~5000r/min；The time of the centrifugation is 12~15min；Step Suddenly temperature dry described in (3) is 40~60 DEG C, dry to constant weight.

A kind of calyx Thick many candies (CAVAPs) from generation to generation, is prepared by above-mentioned preparation method.

The refined polysaccharide of calyx from generation to generation is by the way that the above-mentioned Thick many candies of calyx from generation to generation (CAVAPs) to be further purified to obtain；Institute State from generation to generation calyx refined polysaccharide be six kinds from generation to generation calyx refined polysaccharide (be denoted as CP-I, CP-II, CP-III, CP-IV, CP-V and CP-VI)

The refined polysaccharide of calyx from generation to generation (CP-I) is homogeneous components, and molecular weight (Mn) is 1.3 × 10⁴Da；Specifically For 13169Da；

The refined polysaccharide of calyx from generation to generation (CP-II) is homogeneous components, and molecular weight (Mn) is 7.5 × 10³Da；Specifically For 7538Da；

Described refined polysaccharide of calyx from generation to generation (CP-III, CP-IV, CP-V and CP-VI) the non-homogeneous components.

The refined polysaccharide of calyx from generation to generation (being denoted as CP-I, CP-II, CP-III, CP-IV, CP-V and CP-VI) is by such as Lower step is prepared:

(1) Z-type chromatographic column will be transferred to after 40~60 DEG C of rotary evaporation bubble removings by pretreated DEAE-52 resin In, with constant flow pump pumping distilled water so that filler dress column is uniform, 5~10s/ of flow velocity drop after balance；

(2) polysaccharide solution will be configured to by calyx Thick many candies from generation to generation, be transferred in chromatographic column, and successively use distilled water, 0.05mol/L NaCl, 0.1mol/L NaCl, 0.15mol/L NaCl, 0.2mol/L NaCl and the elution of 0.3mol/L NaCl solution, flow velocity 5 ~10s/ drop collects each fraction respectively, and phend-sulphuric acid tracing detection eluent measures the absorbance under 490nm, and drafting is washed Curve is taken off, the eluent in same absorption peak merges；Then concentrated by rotary evaporation, vacuum freeze drying, by DEAE-52 ion exchange Six components are obtained after column chromatography, wherein the refined polysaccharide that distilled water affords is named as CP-I, and 0.05mol/L NaCl is washed De- obtained refined polysaccharide is named as CP-II, and the refined polysaccharide that 0.1mol/L NaCl is afforded is named as CP-III, The refined polysaccharide that 0.15mol/L NaCl is afforded is named as CP-IV, the refined polysaccharide life that 0.2mol/L NaCl is afforded The refined polysaccharide that entitled CP-V, 0.3mol/L NaCl are afforded is named as CP-VI.

The pretreated method of DEAE-52 resin described in step (1) are as follows: impregnate DEAE-52 resin with 4~30 DEG C of water 12~for 24 hours；0.5~2h is impregnated with the hydrochloric acid solution of 0.5mol/L again；The NaOH solution of 0.5mol/L impregnates 0.5~2h, filters, It is washed to neutrality, is obtained by pretreated DEAE-52 resin；

The temperature of concentrated by rotary evaporation described in step (2) is 40~60 DEG C；

The Thick many candies of calyx from generation to generation or refined polysaccharide are in preparation anticancer, anti-oxidant, in medicament for immunity enhancement application.

The Thick many candies of calyx from generation to generation or refined polysaccharide can be used as novel anticancer, anti-oxidant, medicament for immunity enhancement.

A kind of medicament for immunity enhancement contains the above-mentioned Thick many candies of calyx from generation to generation or refined polysaccharide；

The principle of the present invention: the polysaccharide of plant origin has a variety of biologies such as anti-oxidant, antitumor and enhancing immune function Activity.Polysaccharide especially can embody various beneficial pharmacological actions by adjusting the immune function of macrophage.The present invention passes through Experiment in vitro discovery and confirmation plant Thick many candies or refined polysaccharide have anti-oxidant, antitumor and immune-enhancing activity, and use Dosage is low, and the toxic side effect under same dosage is relatively low.

The present invention has the following advantages and effects with respect to the prior art:

(1) present invention firstly discovers that calyx Thick many candies (CAVAPs) have certain removing and suppression to DPPH free radical from generation to generation Production is used；

(2) present invention firstly discovers that calyx Thick many candies (CAVAPs) are to human breast cancer cell line Bcap-37 and lung carcinoma cell from generation to generation HCC827 has certain inhibited proliferation；

(3) present invention firstly discovers that from generation to generation calyx Thick many candies (CAVAPs) and refined polysaccharide (CP-I, CP-II, CP-III, CP-IV, CP-V and CP-VI) release of RAW264.7 macrophage NO can be significantly increased；

(4) present invention firstly discovers that calyx Thick many candies (CAVAPs) and refined polysaccharide (CP-II) can significantly increase from generation to generation The release of RAW264.7 macrophage IL-6 and TNF-α；

(5) present invention firstly discovers that calyx Thick many candies (CAVAPs) and refined polysaccharide (CP-II) can dramatically increase from generation to generation The expression of RAW264.7 macrophage iNOS, IL-6, TNF-α and IL-1 β mRNA；

(6) present invention firstly discovers that calyx Thick many candies (CAVAPs) and refined polysaccharide (CP-II) can dramatically increase from generation to generation The expression of RAW264.7 macrophage p-ERK, p-JNK, p-P38 and p-P65；

(7) dosage of calyx Thick many candies and refined polysaccharide is relatively low from generation to generation, and within the scope of effective dosage Toxicity is lower.

Detailed description of the invention

Fig. 1 be embodiment 4 be prepared the refined polysaccharide of calyx from generation to generation (CP-I, CP-II, CP-III, CP-IV, CP-V and CP-VI ion-exchange chromatography elution curve)；

Fig. 2 be embodiment 4 be prepared the refined polysaccharide of calyx from generation to generation (CP-I, CP-II, CP-III, CP-IV, CP-V and CP-VI High Performance Gel Permeation chromatography (GPC) figure)；The wherein GPC figure that A is calyx refined polysaccharide CP-I from generation to generation, B is bitter orange flower The GPC of calyx refined polysaccharide CP-II schemes, and the GPC figure that C is calyx refined polysaccharide CP-III from generation to generation, D is calyx refined polysaccharide from generation to generation The GPC of CP-IV schemes, the GPC figure that E is calyx refined polysaccharide CP-V from generation to generation, the GPC figure that F is calyx refined polysaccharide CP-VI from generation to generation；

Fig. 3 is the sephadex Sephadex of the refined polysaccharide of calyx from generation to generation CP-I and CP-II that embodiment 4 is prepared G-100 column chromatography elution curves figure；Wherein A is the gel filtration chromatography elution curve of calyx refined polysaccharide CP-I from generation to generation, and B is generation The gel filtration chromatography elution curve of seville orange flower calyx refined polysaccharide CP-II；

Fig. 4 is the infrared spectrogram of the refined polysaccharide of calyx from generation to generation CP-I and CP-II that embodiment 4 is prepared；Wherein A is The infrared spectrogram of calyx refined polysaccharide CP-I from generation to generation, B are the infrared spectrogram of calyx refined polysaccharide CP-II from generation to generation；

Fig. 5 is curve graph of the Thick many candies of the calyx from generation to generation CAVAPs to DPPH free radical inhibiting rate of the preparation of embodiment 1；

Fig. 6 is proliferation inhibition rate of the Thick many candies of the calyx from generation to generation CAVAPs to human breast cancer cell line Bcap-37 of the preparation of embodiment 1 Curve graph；

Fig. 7 is proliferation inhibition rate of the Thick many candies of the calyx from generation to generation CAVAPs to lung carcinoma cell HCC827 of the preparation of embodiment 1 Curve graph；

Fig. 8 is the Thick many candies of calyx from generation to generation CAVAPs prepared by embodiment 1 and calyx refined polysaccharide from generation to generation prepared by embodiment 4 (CP-I, CP-II, CP-III, CP-IV, CP-V and CP-VI) is to the influence diagram of RAW264.7 macrophage survival rate, wherein A Calyx Thick many candies CAVAPs is to the influence diagram of RAW264.7 macrophage survival rate from generation to generation, B be from generation to generation calyx refined polysaccharide CP-I, The influence diagram of CP-II, CP-III, CP-IV, CP-V and CP-VI to RAW264.7 macrophage survival rate；

Fig. 9 is the Thick many candies of calyx from generation to generation CAVAPs prepared by embodiment 1 and calyx refined polysaccharide from generation to generation prepared by embodiment 4 The influence diagram of (CP-I, CP-II, CP-III, CP-IV, CP-V and CP-VI) to RAW264.7 macrophage release NO；Wherein A is From generation to generation calyx Thick many candies CAVAPs to RAW264.7 macrophage release NO influence diagram, B be from generation to generation calyx refined polysaccharide CP-I, The influence diagram of CP-II, CP-III, CP-IV, CP-V and CP-VI to RAW264.7 macrophage release NO；

Figure 10 is that the Thick many candies of calyx from generation to generation CAVAPs prepared by embodiment 1 and the purification of calyx from generation to generation prepared by embodiment 4 are more Influence diagram of the sugared CP-II to RAW264.7 macrophage release IL-6；Wherein A is CAVAPs pairs of calyx Thick many candies from generation to generation RAW264.7 macrophage discharges the influence diagram of IL-6, and B is that calyx refined polysaccharide CP-II releases RAW264.7 macrophage from generation to generation Put the influence diagram of IL-6；

Figure 11 is that the Thick many candies of calyx from generation to generation CAVAPs prepared by embodiment 1 or the purification of calyx from generation to generation prepared by embodiment 4 are more Influence diagram of the sugared CP-II to RAW264.7 macrophage release TNF-α；Wherein A is that CAVAPs releases RAW264.7 macrophage The influence diagram of TNF-α is put, B is the influence diagram that CP-II discharges TNF-α to RAW264.7 macrophage；

Figure 12 is that the Thick many candies of calyx from generation to generation CAVAPs prepared by embodiment 1 and the purification of calyx from generation to generation prepared by embodiment 4 are more The influence diagram that sugared CP-II expresses RAW264.7 macrophage iNOS mRNA, wherein A is that CAVAPs is thin to RAW264.7 macrophage The influence diagram of born of the same parents iNOS mRNA expression, B are the influence diagram that CP-II expresses RAW264.7 macrophage iNOS mRNA, In, *: compared with control group, P < 0.05；*: compared with control group, P < 0.01；

Figure 13 is that the Thick many candies of calyx from generation to generation CAVAPs prepared by embodiment 1 and the purification of calyx from generation to generation prepared by embodiment 4 are more The influence diagram that sugared CP-II expresses RAW264.7 macrophage IL-6mRNA, wherein A is CAVAPs to RAW264.7 macrophage The influence diagram of IL-6mRNA expression, B are the influence diagram that CP-II expresses RAW264.7 macrophage IL-6mRNA mRNA, In, *: compared with control group, P < 0.05；*: compared with control group, P < 0.01；

Figure 14 is that the Thick many candies of calyx from generation to generation CAVAPs prepared by embodiment 1 and the purification of calyx from generation to generation prepared by embodiment 4 are more The influence diagram that sugared CP-II expresses RAW264.7 macrophage TNF-α mRNA, wherein A is that CAVAPs is thin to RAW264.7 macrophage The influence diagram of born of the same parents' TNF-α mRNA expression, B are the influence diagram that CP-II expresses RAW264.7 macrophage TNF-α mRNA mRNA, Wherein, *: compared with control group, P < 0.05；*: compared with control group, P < 0.01；

Figure 15 is that the Thick many candies of calyx from generation to generation CAVAPs prepared by embodiment 1 and the purification of calyx from generation to generation prepared by embodiment 4 are more The influence diagram that sugared CP-II expresses RAW264.7 macrophage IL-1 β mRNA；Wherein A is that CAVAPs is thin to RAW264.7 macrophage The influence diagram of born of the same parents IL-1 β mRNA expression, B are the influence diagram that CP-II expresses RAW264.7 macrophage IL-1 β mRNA mRNA, Wherein, *: compared with control group, P < 0.05；*: compared with control group, P < 0.01；

Figure 16 is that the Thick many candies of calyx from generation to generation CAVAPs prepared by embodiment 1 and the purification of calyx from generation to generation prepared by embodiment 4 are more Influence diagram of the sugared CP-II to RAW264.7 macrophage p-ERK protein expression；The wherein p-ERK albumen table that figure A-1 is CAVAPs Up to exposure diagram, A-2 is p-ERK albumen relative expression's spirogram of CAVAPs；Scheme the p-ERK protein expression that B-1 is CP-II to expose Figure, B-2 are p-ERK albumen relative expression's spirogram of CP-II, wherein *: compared with control group, P < 0.05；*: with Control group compares, P < 0.01；

Figure 17 is that the Thick many candies of calyx from generation to generation CAVAPs prepared by embodiment 1 and the purification of calyx from generation to generation prepared by embodiment 4 are more Influence diagram of the sugared CP-II to RAW264.7 macrophage p-JNK protein expression；The wherein p-JNK albumen table that figure A-1 is CAVAPs Up to exposure diagram, p-JNK albumen relative expression's spirogram that A-2 is CAVAPs is schemed；Scheme the p-JNK protein expression that B-1 is CP-II to expose Figure, p-JNK albumen relative expression's spirogram that figure B-2 is CP-II, wherein *: compared with control group, P < 0.05；*: with Control group compares, P < 0.01；

Figure 18 is that the Thick many candies of calyx from generation to generation CAVAPs prepared by embodiment 1 and the purification of calyx from generation to generation prepared by embodiment 4 are more Influence diagram of the sugared CP-II to RAW264.7 macrophage p-P38 protein expression；The wherein p-P38 albumen table that figure A-1 is CAVAPs Up to exposure diagram, p-P38 albumen relative expression's spirogram that A-2 is CAVAPs is schemed；Scheme the p-P38 protein expression that B-1 is CP-II to expose Figure, p-P38 albumen relative expression's spirogram that figure B-2 is CP-II, wherein *: compared with control group, P < 0.05；*: with Control group compares, P < 0.01；

Figure 19 is that the Thick many candies of calyx from generation to generation CAVAPs prepared by embodiment 1 and the purification of calyx from generation to generation prepared by embodiment 4 are more Influence diagram of the sugared CP-II to RAW264.7 macrophage p-P65 protein expression；The wherein p-P65 albumen table that figure A-1 is CAVAPs Up to exposure diagram, p-P65 albumen relative expression's spirogram that A-2 is CAVAPs is schemed；Scheme the p-P65 protein expression that B-1 is CP-II to expose Figure, p-P65 albumen relative expression's spirogram that figure B-2 is CP-II, wherein *: compared with control group, P < 0.05；*: with Control group compares, P < 0.01.

Specific embodiment

Below with reference to embodiment and attached drawing, the present invention is described in further detail, but embodiments of the present invention are unlimited In this.In embodiment, human breast cancer cell line Bcap-37, lung carcinoma cell HCC827 and mouse macrophage RAW 264.7 are purchased from middle section Shanghai life science institute, institute cell resource center；Calyx is purchased from the peaceful medicinal material market in Guangzhou from generation to generation, and the place of production is Zhejiang.

Calyx Thick many candies (CAVAPs) from generation to generation is prepared in embodiment 1 from calyx from generation to generation:

(1) calyx smashes it through 20 meshes from generation to generation, weighs 100g, with distilled water heating and refluxing extraction, wherein ratio of water to material is 20:1 (mass ratio), refluxing extraction temperature are 75 DEG C, reflux extracting time 4h, refluxing extraction number 3 times；It is then combined with extraction Liquid is centrifuged 10min under 4000r/min, takes supernatant liquid, and filter paper filter 23 subtracts filtrate with Rotary Evaporators at 40 DEG C Pressure is concentrated into 200mL, obtains polysaccharide solution；

(2) D354FD resin is first used into distilled water immersion 12h, then successively uses hydrochloric acid solution, matter that mass fraction is 5% The sodium hydroxide solution that amount score is 5% respectively impregnates 0.5h, 200 mesh filtered through gauze, and distillation is washed to neutrality, obtains by pre- The D354FD resin of processing；

(3) polysaccharide solution and 200mL step (1) being prepared are mixed by pretreated D354FD resin, are placed in Water bath with thermostatic control 3h decolourizes in 50 DEG C of thermostat water baths, then interval stirring goes out polysaccharide solution with the filter-cloth filtering of 200 mesh, And clean 5 times repeatedly with water and wash out the polysaccharide adsorbed in resin, the polysaccharide liquid after decoloration is merged, with Rotary Evaporators at 40 DEG C Under be concentrated under reduced pressure into 200mL, obtain concentration liquid glucose；

(4) dehydrated alcohol of concentration 4 times of volumes of liquid glucose, side edged are added in the concentration liquid glucose that step (3) is prepared Magnetic agitation, the alcohol precipitation 8h under the conditions of 4 DEG C, is then centrifugated 15min under 4000rpm, discards supernatant liquid, take out sediment in 45 DEG C of drying, obtain calyx Thick many candies (CAVAPs) from generation to generation.

Calyx Thick many candies (CAVAPs) from generation to generation is prepared in embodiment 2 from calyx from generation to generation:

(1) calyx smashes it through 20 meshes from generation to generation, weighs 100g, then distilled water heating and refluxing extraction, wherein ratio of water to material 20:1 (mass ratio), refluxing extraction temperature are 85 DEG C, reflux extracting time 3h, refluxing extraction number 2 times；It is then combined with extraction Liquid is centrifuged 8min under 3000r/min, takes supernatant liquid, and filter paper filters 2 times, filtrate is subtracted at 50 DEG C with Rotary Evaporators Pressure is concentrated into 200mL, obtains polysaccharide solution；

(2) by D354FD resin first with distilled water immersion for 24 hours, then successively with mass fraction be 5% hydrochloric acid solution, matter The sodium hydroxide solution that amount score is 5% respectively impregnates 2h, 200 mesh filtered through gauze, and distillation is washed to neutrality, obtains by locating in advance The D354FD resin of reason；

(3) polysaccharide solution and 200mL step (1) being prepared are mixed by pretreated D354FD resin, are placed in Water bath with thermostatic control 2h decolourizes in 40 DEG C of thermostat water baths, then interval stirring goes out polysaccharide solution with the filter-cloth filtering of 200 mesh, And clean 4 times repeatedly with water and wash out the polysaccharide adsorbed in resin, the polysaccharide liquid after decoloration is merged, with Rotary Evaporators at 50 DEG C Under be concentrated under reduced pressure into 200mL, obtain concentration liquid glucose；

(4) ethyl alcohol that the volume fraction of 3 times of volumes is 95% is added in the concentration liquid glucose being prepared in step (3), Side edged magnetic agitation, the alcohol precipitation 12h under the conditions of 0 DEG C, is then centrifugated 12min under 3000rpm, discards supernatant liquid, takes out Sediment is dried in 50 DEG C, obtains calyx Thick many candies (CAVAPs) from generation to generation.

Calyx Thick many candies (CAVAPs) from generation to generation is prepared in embodiment 3 from calyx from generation to generation:

(1) calyx smashes it through 20 meshes from generation to generation, weighs 100g, with distilled water heating and refluxing extraction, wherein ratio of water to material is 20:1 (mass ratio), refluxing extraction temperature are 95 DEG C, reflux extracting time 2h, refluxing extraction number 4 times, are then combined with extraction Liquid is centrifuged 12min under 5000/min, takes supernatant liquid, and filter paper filters 4 times, filtrate is subtracted at 60 DEG C with Rotary Evaporators Pressure is concentrated into 200mL, obtains polysaccharide solution；

(2) D354FD resin is first used into distilled water immersion 18h, then successively uses hydrochloric acid solution, matter that mass fraction is 5% The sodium hydroxide solution that amount score is 5% respectively impregnates 3h, 200 mesh filtered through gauze, and distillation is washed to neutrality, obtains by locating in advance The D354FD resin of reason；

(3) polysaccharide solution and 200mL that are prepared in step (1) are mixed by pretreated D354FD resin, is set Water bath with thermostatic control 4h decolourizes in 60 DEG C of thermostat water baths, and it is molten to go out polysaccharide with the filter-cloth filtering of 200 mesh later for interval stirring Liquid, and clean 5 times repeatedly with water and wash out the polysaccharide adsorbed in resin, the polysaccharide liquid after decoloration is merged, is existed with Rotary Evaporators It is concentrated under reduced pressure into 200mL at 60 DEG C, obtains concentration liquid glucose；

(4) ethyl alcohol that the volume fraction of 5 times of volumes is 95%, side is added in the concentration liquid glucose that step (3) is prepared Edged magnetic agitation, alcohol precipitation for 24 hours, is then centrifugated 14min under 5000rpm under the conditions of 4 DEG C, discards supernatant liquid, and it is heavy to take out Starch is dried in 60 DEG C, obtains calyx Thick many candies (CAVAPs) from generation to generation.

The preparation of the calyx refined polysaccharide (CP-I, CP-II, CP-III, CP-IV, CP-V and CP-VI) from generation to generation of embodiment 4:

(1) DEAE-52 is pre-processed: being taken the DEAE-52 of 70g, is impregnated for 24 hours with 4 DEG C of distilled water, then carefully fall upper water Out, impurity is removed, then impregnates 0.5h with the hydrochloric acid solution of 0.5mol/L, upper layer acid solution is carefully poured out, filters to after doing to use and steam Distilled water is eluted to neutrality, then impregnates 0.5h with the NaOH solution of 0.5mol/L, carefully pours out upper layer lye, filters to after doing with steaming Distilled water is washed till neutrality, spare；

(2) glass bar drainage 2.6 × 30cm will be transferred to after pretreated DEAE-52 is in 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 dress column is uniform, 7s/ drop after balance；

(3) Thick many candies of calyx from generation to generation (CAVAPs) being prepared in 100mg embodiment 1 are weighed, polysaccharide solution is configured to, Glass bar is drained in chromatographic column, successively uses distilled water, 0.05mol/L NaCl, 0.1mol/L NaCl, 0.15mol/L NaCl, 0.2mol/L NaCl and the elution of 0.3mol/L NaCl solution, collect each fraction automatically, and every root canal collects about 5mL, benzene Phenol-sulfuric acid method tracing detection eluent measures the absorbance at 490nm, draws elution curve, the eluent in same absorption peak Merge；Then 40 DEG C of concentrated by rotary evaporations of eluent, vacuum freeze drying；CAVAPs is obtained after DEAE-52 ion-exchange chromatography To six components, wherein the refined polysaccharide that distilled water affords is named as CP-I, the essence that 0.05mol/L NaCl is afforded Polysaccharide processed is named as CP-II, and the refined polysaccharide of calyx from generation to generation that 0.1mol/L NaCl is afforded is named as CP-III, The refined polysaccharide that 0.15mol/L NaCl is afforded is named as CP-IV, the refined polysaccharide life that 0.2mol/L NaCl is afforded The refined polysaccharide that entitled CP-V, 0.3mol/L NaCl are afforded is named as CP-VI, wherein Fig. 1 is the ion of the present embodiment Exchange column chromatography elution curves figure.

The preparation of the calyx refined polysaccharide (CP-I, CP-II, CP-III, CP-IV, CP-V and CP-VI) from generation to generation of embodiment 5:

(1) DEAE-52 is pre-processed: the DEAE-52 of 70g is taken, with 20 DEG C of immersion 12h of distilled water, then carefully by upper water It pours out, removes impurity, then impregnate 1h with the hydrochloric acid solution of 0.5mol/L, upper layer acid solution is carefully poured out, filter to after doing to use and steam Distilled water is eluted to neutrality, then impregnates 1h with the NaOH solution of 0.5mol/L, carefully pours out upper layer lye, filters to after doing with distillation It is washed to neutrality, it is spare；

(3) Thick many candies of calyx from generation to generation (CAVAPs) being prepared in 100mg embodiment 1 are weighed, polysaccharide solution is configured to, Glass bar is drained in chromatographic column, successively uses distilled water, 0.05mol/L NaCl, 0.1mol/L NaCl, 0.15mol/L NaCl, 0.2mol/L NaCl and the elution of 0.3mol/L NaCl solution, collect each fraction automatically, and every root canal collects about 5mL, benzene Phenol-sulfuric acid method tracing detection eluent measures the absorbance at 490nm, draws elution curve, the eluent in same absorption peak Merge；Then 50 DEG C of concentrated by rotary evaporations of eluent, vacuum freeze drying；CAVAPs is obtained after DEAE-52 ion-exchange chromatography To six components, wherein the refined polysaccharide that distilled water affords is named as CP-I, the essence that 0.05mol/L NaCl is afforded Polysaccharide processed is named as CP-II, and the refined polysaccharide that 0.1mol/L NaCl is afforded is named as CP-III, 0.15mol/L NaCl The refined polysaccharide afforded is named as CP-IV, and the refined polysaccharide that 0.2mol/L NaCl is afforded is named as CP-V, The refined polysaccharide that 0.3mol/L NaCl is afforded is named as CP-VI, wherein the ion-exchange chromatography of the present embodiment elutes As a result with embodiment 4.

The preparation of the calyx refined polysaccharide (CP-I, CP-II, CP-III, CP-IV, CP-V and CP-VI) from generation to generation of embodiment 6:

(1) DEAE-52 is pre-processed: the DEAE-52 of 70g is taken, with 25 DEG C of immersion 20h of distilled water, then carefully by upper water It pours out, removes impurity, then impregnate 0.8h with the hydrochloric acid solution of 0.5mol/L, upper layer acid solution is carefully poured out, filters to after doing and uses Distilled water is eluted to neutrality, then impregnates 0.8h with the NaOH solution of 0.5mol/L, carefully pours out upper layer lye, filters to after doing and use Distillation is washed to neutrality, spare；

(3) Thick many candies of calyx from generation to generation (CAVAPs) being prepared in 100mg embodiment 1 are weighed, polysaccharide solution is configured to, Glass bar is drained in chromatographic column, successively uses distilled water, 0.05mol/L NaCl, 0.1mol/L NaCl, 0.15mol/L NaCl, 0.2mol/L NaCl and the elution of 0.3mol/L NaCl solution, collect each fraction automatically, and every root canal collects about 5mL, benzene Phenol-sulfuric acid method tracing detection eluent measures the absorbance at 490nm, draws the eluent in the same absorption peak of elution curve Merge；Then 60 DEG C of concentrated by rotary evaporations of eluent, vacuum freeze drying；CAVAPs is obtained after DEAE-52 ion-exchange chromatography To six components, wherein the refined polysaccharide that distilled water affords is named as CP-I, the essence that 0.05mol/L NaCl is afforded Polysaccharide processed is named as CP-II, and the refined polysaccharide that 0.1mol/L NaCl is afforded is named as CP-III, 0.15mol/L NaCl The refined polysaccharide afforded is named as CP-IV, and the refined polysaccharide that 0.2mol/L NaCl is afforded is named as CP-V, The refined polysaccharide that 0.3mol/L NaCl is afforded is named as CP-VI.Wherein, the ion-exchange chromatography elution of the present embodiment As a result with embodiment 4.

The gpc analysis of embodiment 7 calyx refined polysaccharide CP-I, CP-II, CP-III, CP-IV, CP-V and CP-VI from generation to generation

Embodiment 4~6 is prepared respectively using high performance liquid chromatograph CP-I, CP-II, CP-III, CP-IV, The molecular weight and its purity of CP-V and CP-VI is measured.Chromatographic condition: TSK G-5000PXL column (7.8 × 300mm) It connects with TSK G-3000PXL column (7.8 × 300mm), mobile phase is the KH of 0.02mol/L₂PO₄Buffer solution, flow velocity For 0.6mL/min, 2414 Composition distributions, 35 DEG C of column temperature.Fig. 2 is the calyx refined polysaccharide from generation to generation that embodiment 4 is prepared High Performance Gel Permeation chromatography (GPC) figure of (CP-I, CP-II, CP-III, CP-IV, CP-V and CP-VI).

Interpretation of result:

(1) the GPC figure of embodiment 4 is prepared CP-I (Fig. 2A) and CP-II (Fig. 2 B) are single symmetrical peak, are shown CP-I and CP-II is homogeneous polysaccharide, and molecular weight (Mn) is respectively 13169Da and 7538Da.What embodiment 4 was prepared CP-III (Fig. 2 C), CP-IV (Fig. 2 D), CP-V (Fig. 2 E) and CP-VI (Fig. 2 F) GPC figure be not single symmetrical peak, show The non-homogeneous components of CP-III, CP-IV, CP-V and CP-VI.The result of embodiment 5 and 6 is the same as embodiment 4.

The Sephadex of embodiment 8 calyx refined polysaccharide CP-I, CP-II, CP-III, CP-IV, CP-V and CP-VI from generation to generation G-100 column chromatographic analysis

Sephadex G-100 pre-treatment: taking a certain amount of Sephadex G-100 in the beaker of 500m L, is added and steams Distilled water 15~20m L/g, the continuous heating 5h in 90 DEG C of thermostat water baths, the careful stirring of interval, after it is cooled to room temperature to With.

The Sephadex G-100 certain volume of the above method of learning from else's experience processing, glass bar is drained to glass bar after mixing evenly In the Z-type chromatographic column of 2.6 × 30cm, being pumped into distilled water with constant flow pump keeps the dress column in chromatographic column uniform, adjusts flow velocity to balance Flow velocity is 15s/ drop afterwards.Each 10mg of CP-I and CP-II is weighed, the polysaccharide solution of 2mg/m L is configured to, through sephadex Sephadex G-100 column chromatography, 12min/ pipe, with distillation water elution, phend-sulphuric acid tracing detection draws elution curve simultaneously It is collected according to peak.Fig. 3 is the sephadex of the refined polysaccharide of calyx from generation to generation CP-I and CP-II that embodiment 4 is prepared Sephadex G-100 column chromatography elution curves figure；The gel filtration chromatography that wherein A is calyx refined polysaccharide CP-I from generation to generation elutes bent Line chart, B are the gel filtration chromatography elution curve of calyx refined polysaccharide CP-II from generation to generation.

Interpretation of result:

The CP-I (Fig. 3 A) and CP-II (Fig. 3 B) sephadex Sephadex G-100 gel that embodiment 4 is prepared Column chromatography, the elution curve that phend-sulphuric acid tracing detection obtains is single symmetrical peak, with High Performance Gel Permeation chromatography one It causes, further confirms that CP-I and CP-II is homogeneous polysaccharide.The result of embodiment 5 and 6 is the same as embodiment 4.

9 IR spectrum scanning of embodiment

The refined polysaccharide of calyx from generation to generation (the CP-I and CP-II) 2mg being prepared in Example 4~6 respectively, it is mixed with KBr Close it is finely ground at thin uniform layer shape, in 4000~400cm^-1Infrared scan is carried out in wave-number range.Fig. 4 is that embodiment 4 is prepared The refined polysaccharide of calyx from generation to generation CP-I and CP-II infrared spectrogram；Wherein A is the infrared light of calyx refined polysaccharide CP-I from generation to generation Spectrogram, B are the infrared spectrogram of calyx refined polysaccharide CP-II from generation to generation.

Interpretation of result:

Polysaccharide results of IR:

The strong absworption peak of CP-I (Fig. 4 A) and CP-II (Fig. 4 B) successively at wave number 3432cm-1 and 3448cm-1 is carbohydrate Intermolecular or intramolecular O-H stretching vibration peak, shape are wide and blunt, it is known that hydroxyl associates intermolecular, is not free hydroxyl Base.CP-I and CP-II is in the C-H absorption of vibrations that the absorption peak of wave number 2896cm-1 and 2912cm-1 everywhere is methine or methyl Peak, caused by 1450~1250cm-1 absorption peak may be the vibration of carbohydrate molecule C-H angle.By the feature of above three groups of carbohydrates Peak can tentatively judge CP-I and CP-II for polysaccharide.CP-I and CP-II has stronger absorption peak at 1643cm-1, shows CP- Contain-COO group in I and CP-II.Absorption peak of two kinds of polysaccharide at 1300-1000cm-1 indicates CP-I and CP-II simultaneously In saccharide ring be configured as pyranoid form (furan type saccharide ring on this section only there are two strong absworption peak), because CP-I and CP-II is pyrrole It mutters ring structure.The CP-I and CP-II absorption peak at 893cm-1 and 851cm-1 respectively both shows mainly with β-D grape pyrrole It mutters based on sugar.The testing result of embodiment 5 and embodiment 6 is the same as embodiment 4.

Inhibiting effect of the calyx Thick many candies (CAVAPs) to DPPH free radical from generation to generation of embodiment 8

The production of standard curve: accurately weighing 19.7mg DPPH and first dissolved with a small amount of dehydrated alcohol, then fixed with dehydrated alcohol Hold to 250ml, be made into the standard solution of 200 μm of ol/L, sets in brown bottle in being saved in refrigerator.Taking 1.5ml concentration respectively is 0, After 10,20,50,100,150,200 μm of ol/L DPPH standard solution and 0.5ml dehydrated alcohol mix, 30min is stood, then Light absorption value is measured at maximum absorption wavelength 517nm.Using concentration as abscissa, absorbance value is that ordinate makes DPPH standard Curve selects suitable DPPH concentration as the DPPH standard solution experimental concentration in measurement sample.

Configure the sample (Thick many candies of calyx from generation to generation (CAVAPs) that embodiment 1 is prepared) and standard items of various concentration (ascorbic acid VC) solution takes 0.5ml sample or standard solution respectively, and the selected suitable concentration of 1.5ml is added DPPH titer (ready-to-use) replaces sample as blank control, replaces DPPH solution with dehydrated alcohol using dehydrated alcohol As Background control, 30min is stood after mixed solution is shaken up, measures light absorption value at 517nm wavelength with cuvette.Sample pair The clearance rate of DPPH free radical is calculated as follows:

DPPH clearance rate (%)=[1- (A sample-A Background control)/A blank control] × 100%.

Test results are shown in figure 5, and Fig. 5 is that the Thick many candies of calyx from generation to generation (CAVAPs) prepared by embodiment 1 are free to DPPH The curve graph of base inhibiting rate.The Thick many candies of calyx from generation to generation (CAVAPs) being prepared as the result is shown by embodiment 1 are to DPPH freedom Base has faint removing and inhibiting effect, but not significant (Fig. 5).

Increasing of the calyx Thick many candies (CAVAPs) to human breast cancer cell line Bcap-37 and lung carcinoma cell HCC827 from generation to generation of embodiment 9 Grow inhibiting effect

By human breast cancer cell line Bcap-37 and lung carcinoma cell HCC827 cell with 1 × 10⁵A/that ml concentration is inoculated in 96 holes is thin Born of the same parents' culture plate (100 hole μ L/), sets 37 DEG C, cultivates in 5% carbon dioxide incubator.After the cell in orifice plate is completely adherent, add Enter the 100 μ l of sample solution of various concentration (62.5,125,250,500 and 1000 μ g/m L).Cell controls group is set simultaneously, i.e., The complete culture solution with sample liquid equivalent volumes is added；Positive controls add the five-fluorouracils with sample with concentration same volume As positive control.Each concentration sets 6 in parallel.At 37 DEG C, 5%CO₂Continue to cultivate in incubator, take out afterwards for 24 hours, is inverted aobvious The variation of micro mirror observation cellular morphology.It is carefully inhaled with syringe and abandons culture solution in 96 orifice plates, washed with the PBS without calcium and magnesium ion Plate 2 times, the 20 μ L of MTT solution (need to turn off the light operation, the light-exposed easy decomposition of MTT) and DMEM that then 5mg/m L is added in every hole are trained completely 180 μ L of nutrient solution, at 37 DEG C, 5%CO₂Continue to cultivate 4h in incubator.Careful to inhale the cell culture fluid abandoned in hole, every hole is added 150 μ L DMSO vibrate 10min.Its absorbance value is measured in the case where wavelength is 490nm with enzyme-linked immunosorbent assay instrument.

The increment inhibiting rate of cancer cell=(blank control group absorbance value-sample sets absorbance value)/blank control group Light absorption value.

Test result is as shown in Figures 6 and 7；Fig. 6 is the Thick many candies of calyx from generation to generation (CAVAPs) of the preparation of embodiment 1 to human milk gland The curve graph of the proliferation inhibition rate of cancer cell MCF-7；Fig. 7 is the Thick many candies of calyx from generation to generation (CAVAPs) of the preparation of embodiment 1 to lung The curve graph of the proliferation inhibition rate of cancer cell HCC827.

The Thick many candies of calyx from generation to generation (CAVAPs) being prepared by embodiment 1 are thin to MCF-7 (Fig. 6) and HCC827 (Fig. 7) Born of the same parents have a degree of inhibiting effect, and stronger for the inhibited proliferation of MCF-7.

Embodiment 10 calyx essence polysaccharide CAVAPs and CP-I, CP-II, CP-III, CP-IV, CIP-V, CP-VI couple from generation to generation The influence of RAW264.7 cell survival rate

By mouse monokaryon macrophage RAW264.7 with 1 × 10⁶A/ml concentration is inoculated in 96 porocyte culture plates (100 μ The hole L/), 37 DEG C are set, 5%CO₂It is cultivated in incubator.After the cell in orifice plate is completely adherent, 100 μ L various concentrations are added CAVAPs (62.5,125,250,400 and 500 μ g/m L) and CP-I, CP-II, CP-III, CP-IV, CIP-V, CP-VI (25, 50,100,150,200 and 250 μ g/m L), it is parallel that each concentration sets 6, while setting cell blank control group.At 37 DEG C, 5% Continue to cultivate in CO2 incubator, take out afterwards for 24 hours, the metamorphosis of cell is observed under inverted microscope.Abandoning is carefully inhaled with syringe Culture supernatants in hole are cleaned twice with PBS, then carry out cell survival rate detection with mtt assay, i.e., every hole is added 5mg/m L's 20 μ L and DMEM complete culture solution of MTT solution, 180 μ L, at 37 DEG C, 5%CO₂Continue to cultivate 4h in incubator.Then careful inhale is abandoned 150 μ L DMSO are added in supernatant in hole, every hole, vibrate 10min.It is measured with enzyme-linked immunosorbent assay instrument in the case where wavelength is 490nm Its absorbance value calculates the survival rate of cell according to following formula:

Opposite proliferation degree (PGR)=experimental group mean absorbance values/blank control group absorbance value × 100%.

Test results are shown in figure 8；Fig. 8 be embodiment 1 prepare CAVAPs and embodiment 4 prepare CP-I, CP-II, The influence diagram of CP-III, CP-IV, CIP-V, CP-VI to RAW264.7 macrophage survival rate.

The CAVAPs being prepared by embodiment 1 is not apparent to RAW264.7 macrophage in 62.5~500 μ g/mL Toxic effect (Fig. 8 A), can be used for next step experimental study；CP-I, CP-II for being prepared by embodiment 4, CP-III, CP-IV, CIP-V and CP-VI does not have apparent toxic effect (Fig. 8 B) to RAW264.7 macrophage in 25~250 μ g/mL, under can be used for One step experimental study.

Embodiment 11Griess method detects CAVAPs and CP-I, CP-II, CP-III, CP-IV, CIP-V, CP-VI couple The influence of RAW264.7 cell release NO

Routine culture cell, the RAW264.7 cell of logarithmic growth phase, piping and druming cell is at 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 a/hole is inoculated in 24 well culture plates, is placed in 37 DEG C, 5%CO₂Incubator culture, adherent supernatant is abandoned in suction for 24 hours, is pressed Following grouping requires administration, and every group sets 3 multiple holes: Control group, lipopolysaccharides (LPS, final concentration of 1 μ g/mL) group, polysaccharide sample Product group (CAVAPs (the μ g/mL of final concentration of 16.13,31.25,62.5,125,250 and 500) group that embodiment 1 is prepared, it is more Sugared sample sets (CP-I, CP-II, CP-III, CP-IV, CIP-V, CP-VI that embodiment 4 is prepared (final concentration of 16.13, 31.25,62.5,125,200 and 250 μ g/mL) group, the culture medium of control group addition same volume.After administration, it is placed in 37 DEG C, 5% CO₂Incubator continues culture for 24 hours.Each 100 μ L of hole cell conditioned medium is taken respectively, is accordingly added in another 96 new orifice plates.Every hole adds 50 μ L Griess reagent As and 50 μ L Griess reagent B, are subsequently placed in 37 DEG C of incubators and react 10min, set plate immediately in more It marks on micropore board detector, each hole absorbance value is detected under 550nm wavelength.

Test results are shown in figure 9；Fig. 9 A is that CAVAPs prepared by embodiment 1 discharges NO to RAW264.7 macrophage Influence diagram, Fig. 9 B are that CP-I, CP-II, CP-III, CP-IV, CIP-V, CP-VI prepared by embodiment 4 are thin to RAW264.7 macrophage The influence diagram of born of the same parents' release NO.CAVAPs and CP-I, CP-II, CP-III, CP-IV, CIP-V, CP-VI can be remarkably promoted The release of RAW264.7 macrophage NO, especially CP-II effect are best.Comprehensively consider, CAVAPs and CP-II is selected in follow-up test Further research.

Embodiment 12Elisa method detects the influence that CAVAPs and CP-II discharges IL-6 and TNF-α to RAW264.7 cell

Routine culture cell, the RAW264.7 cell of logarithmic growth phase, piping and druming cell is at 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 a/hole is inoculated in 24 well culture plates, is placed in 37 DEG C, 5%CO₂Incubator culture, adherent supernatant is abandoned in suction for 24 hours, is pressed Following grouping requires administration, and every group sets 3 multiple holes: Control group, lipopolysaccharides (LPS, final concentration of 1 μ g/mL) group, CAVAPs Group (CAVAPs that embodiment 1 is prepared, final concentration of 31.25,62.5,125,250,500 μ g/mL), CP-II group (are implemented The CP-II that example 4 is prepared, final concentration of 31.25,62.5,125,200,250 μ g/mL), control group adds the training of same volume Support base.After administration, 37 DEG C are placed in, 5%CO₂Incubator continues culture for 24 hours.Take each hole cell conditioned medium according to mouse IL- respectively The release conditions of 6ELISA kit and mouse TNF-α ELISA kit operating method detection cell factor.

Test result is as shown in Figure 10~11；Figure 10 A is that CAVAPs prepared by embodiment 1 releases RAW264.7 macrophage Put the influence diagram of IL-6；Figure 10 B is the influence diagram that CP-II prepared by embodiment 4 discharges IL-6 to RAW264.7 macrophage；Figure 11A is the influence diagram that CAVAPs prepared by embodiment 1 discharges TNF-α to RAW264.7 macrophage；Figure 11 B is that embodiment 4 is made Influence diagram of the standby CP-II to RAW264.7 macrophage release TNF-α；

CAVAPs and CP-II can remarkably promote RAW264.7 macrophage IL-6 and TNF-α release (Figure 10~ 11)。

The shadow that embodiment 13CAVAPs and CP-II expresses RAW264.7 cell iNOS, IL-6, TNF-α and IL-1 β mRNA It rings

Routine culture cell takes the cell to the growth number phase, is inoculated in 6 orifice plates by 1,000,000/hole cell number, is incubated for for 24 hours Afterwards, it inhales and abandons supernatant, by following grouping requirement administration, every group sets 3 multiple holes.Control group, lipopolysaccharides (LPS, final concentration of 1 μ G/mL) group, CAVAPs group (the CAV APs, final concentration of 31.25,62.5,125,250,500 μ g/ that embodiment 1 is prepared ML), CP-II group (CP-II that embodiment 4 is prepared, final concentration of 31.25,62.5,125,200,250 μ g/mL), control The culture medium of group addition same volume.After administration, 37 DEG C are placed in, 5%CO₂Incubator continues culture for 24 hours.After effect for 24 hours, inhales and abandon Cell conditioned medium, PBS are washed 2 times.Trizol 1mL is added in every hole, stands 5min, and suction pipe is blown and beaten to liquid without dope, draws cell Lysate is transferred to EP pipe, and every pipe is added 0.2mL chloroform, covers EP pipe lid, holds and firmly shakes 10s up and down, is stored at room temperature 5min, 4 DEG C are centrifuged 15min with 12,000r/min, and upper strata aqueous phase is shifted after centrifugation into another EP pipe, 0.5mL isopropanol, room temperature is added 10min is stood, 4 DEG C of centrifuges are centrifuged 10min with 12,000r/min, and careful inhale discards supernatant, and clean 2 with cold 75% ethyl alcohol 1mL It is secondary, 5min is centrifuged with 7,500r/min under the conditions of 4 DEG C of difference, careful inhale abandons supernatant, air blow drying, about 15min, 0~50 μ of addition L RNase-free pure water, 60 DEG C of heating 10min dissolution precipitatings.Measure the purity and concentration of mRNA.And use RevertAid First Strand cnthesis Kit (Thermo company) reverse transcription reagent box, 20 μ L reaction systems reverse RNA Record.Using D yNAmo Flash SYRB Green qPCR Kit (Thermo company) kit, ABI real-time fluorescence quantitative PCR Instrument (Rrism 7500, Applied Biosystems, Foster City, CA, USA) expands mRN A, uses after amplification Relative Quantification (d dCt) Study method is automatically analyzed to obtain in ABI PRISM 7500SDS software Obtain the relative expression quantity of target gene.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 - the GCACATCAAAGCGGCCATAG-3 ' of ACATGACTTCAGGC-3 ', reverse, 5 ') and TNF-α (forward, 5 '- GGGGATTATGGCTCAGGGTC-3′,reverse,5′-CGAGGCTCCAGTGAATTCGG-3′)。

Test result is as shown in Figure 12~15.Figure 12 is prepared by CAVAPs (Figure 12 A) prepared by embodiment 1 and embodiment 4 CP-II (Figure 12 B) influence diagram that RAW264.7 macrophage iNOS mRNA is expressed, Figure 13 is prepared by embodiment 1 The influence that CP-II (Figure 13 B) prepared by CAVAPs (Figure 13 A) and embodiment 4 expresses RAW264.7 macrophage IL-6mRNA Figure, Figure 14 are CAVAPs (Figure 14 A) prepared by embodiment 1 and CP-II (Figure 14 B) prepared by embodiment 4 to RAW264.7 macrophage The influence diagram of cell TNF-α mRNA expression, Figure 15 are CAVAPs (Figure 15 A) prepared by embodiment 1 and CP- prepared by embodiment 4 The influence diagram that II (Figure 15 B) expresses RAW264.7 macrophage IL-1 β mRNA, wherein *: compared with control group, P < 0.05；*: compared with control group, P < 0.01.

Compared with control group, CAVAPs and CP-II can remarkably promote iNOS (Figure 12), IL-6 (Figure 13), TNF-α The secretion (P < 0.05) of (Figure 14) and IL-1 β (Figure 15), and gradient dependence is presented.

Embodiment 14CAVAPs and CP-II is to RAW264.7 cell p-ERK, p-JNK, P-p38 and p-P65 protein expression It influences

Routine culture cell takes the RAW264.7 cell to the growth number phase, is inoculated in 6 orifice plates by 1,000,000/hole cell number, It after being incubated for for 24 hours, inhales and abandons supernatant, by following grouping requirement administration, every group sets 3 multiple holes.Control group, and lipopolysaccharides (LPS, it is dense eventually Degree is 1 μ g/mL) group, CAVAPs group (CAVAPs that embodiment 1 is prepared, final concentration of 125,250,500 μ g/mL), CP- II group (CP-II that embodiment 4 is prepared, final concentration of 125,200,250 μ g/mL), control group add the culture of same volume Base.After administration, 37 DEG C are placed in, 5%CO₂Incubator continues culture for 24 hours.After for 24 hours, inhale abandon cell conditioned medium, and be pre-chilled PBS is cleaned twice, collects cell into 1.5mL centrifuge tube, PIPA lysate (the phosphoric acid enzyme inhibitor of 60 μ L is added (PhosSTOP, Roche) and protease inhibitors (cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpack, Roche)), lysate is drawn with the syringe of 1mL and blows and beats cell repeatedly to mixing well, and is cracked on ice 40min, and every 10min in vibrating 15-20s on turbula shaker.In 4 DEG C, 12000rpm is centrifuged 20min, and supernatant is shifted Managed to new 1.5mL EP, then using BCA method detection protein concentration (according to BCA Protein Assay Kit specification into Row): first by concentration be 2mg/mL BSA solution give ultra-pure deionized water be diluted to series mass concentration (0 μ g/mL, 25 μ g/mL, 125 μ g/mL, 250 μ g/mL, 500 μ g/mL, 1000 μ g/mL) standard solution, further according to needing to configure a certain amount of work Liquid (BCA Solution:4% (volume fraction) Cupric Sulfate=200:4).Take one piece of 96 orifice plate without substrate, every hole It is separately added into the 25 μ L of serial standards solution diluted, and the 25 μ L of protein solution (having diluted 10 times) for needing to detect, Every group is respectively provided with multiple holes.200 hole μ L/ working solutions are added simultaneously, gently concussion mixes, after being placed in 37 DEG C of incubation 30min, take out, OD value is detected at microplate reader 570nm.Standard curve is drawn according to the surveyed OD value of standard items protein solution, calculates testing protein Solution concentration.According to the protein concentration that BCA method measures, albumen volume needed for calculating 40 μ g total proteins, while add 5 μ L 5 × SDS-PAGE sample-loading buffer, ultra-pure deionized water to the total volume for being supplemented respective volume is 25 μ L, mixes, is sufficiently centrifuged, Make liquid accumulation in bottom, 100 DEG C of metal bath heat denatured 10min are centrifuged, are placed in spare on ice.(the volume point of selection 10% Number) glue is concentrated in separation gel and 5% (volume fraction), the protein sample being denaturalized is sequentially added into each swimming lane, sample two sides Swimming lane is separately added into 5 μ L Marker.The TGS buffer of sufficient amount, deposition condition are added in electrophoresis tank are as follows: 100V, about 150min, until stopping electrophoresis when bromophenol blue indicator is run to away from glue lower edge about 0.5cm.Then with the electricity of 100V in ice bath Press transferring film 100min.It is closed with the TBST containing 5% (mass fraction) skimmed milk power, slowly sways 1h.One antiantibody is respectively as follows: GAPDH (14c10) Rabbit mAb, pERK p-JNK, p-P38 and NF- Κ b p-P65 (being purchased from CST company), two antiantibodys For goat antirabbit (HRP label), it is purchased from Ju Yan Biotechnology Co., Ltd.Recommend to specifications dilution ratio (1: 1000) primary antibody, is prepared, jog is incubated for 4h or 4 DEG C and stands overnight at room temperature.After primary antibody is incubated for, secondary antibody, HRP label are replaced Secondary antibody by corresponding proportion dilution (1:2000), room temperature jog 1h.After secondary antibody, it is (public purchased from BioFuture that luminescent solution is added Department) and developed using gel imager protein band.

Figure 16 is CAVAPs prepared by embodiment 1 and CP-II prepared by embodiment 4 to RAW264.7 macrophage p-ERK The influence diagram of protein expression, Figure 16 A-1 are the p-ERK protein expression exposure diagram of CAVAPs prepared by embodiment 1, and 16A-2 is real The p-ERK albumen relative expression quantity of the CAVAPs of the preparation of example 1 is applied, Figure 16 B-1 is the p-ERK albumen of CP-II prepared by embodiment 4 Exposure diagram is expressed, 16B-2 is the p-ERK albumen relative expression quantity of CP-II prepared by embodiment 4；Figure 17 is prepared by embodiment 1 For CP-II prepared by CAVAPs and embodiment 4 to the influence diagram of RAW264.7 macrophage p-JNK protein expression, Figure 17 A-1 is real The p-JNK protein expression exposure diagram of the CAVAPs of the preparation of example 1 is applied, 17A-2 is the p-JNK albumen of CAVAPs prepared by embodiment 1 Relative expression quantity, Figure 17 B-1 are the p-JNK protein expression exposure diagram of CP-II prepared by embodiment 4, and 17B-2 is the system of embodiment 4 The p-JNK albumen relative expression quantity of standby CP-II；Figure 18 is CAVAPs prepared by embodiment 1 and CP-II prepared by embodiment 4 To the influence diagram of RAW264.7 macrophage p-P38 protein expression, Figure 18 A-1 is the p-P38 egg of CAVAPs prepared by embodiment 1 White expression exposure diagram, 18A-2 are the p-P38 albumen relative expression quantity of CAVAPs prepared by embodiment 1, and Figure 18 B-1 is embodiment 4 The p-P38 protein expression exposure diagram of the CP-II of preparation, 18B-2 are the p-P38 albumen relative expression of CP-II prepared by embodiment 4 Amount；Figure 19 is CAVAPs prepared by embodiment 1 and CP-II prepared by embodiment 4 to RAW264.7 macrophage p-P65 albumen table The influence diagram reached, Figure 19 A-1 are p-P65 protein expression exposure diagram, and 19A-2 is p-P65 albumen relative expression quantity, and Figure 19 B-1 is The p-P65 protein expression exposure diagram of CP-II prepared by embodiment 4,19B-2 are the p-P65 albumen of CP-II prepared by embodiment 4 Relative expression quantity.Wherein, *: compared with control group, P < 0.05；*: compared with control group, P < 0.01.

ERK, JNK, P38 and P65 protein expression of phosphorylation are lower in normal group RAW264.7 macrophage, when different dense CAVAPs (final concentration of 125,250,500 μ g/mL), the CP-II (final concentration of 125,200,250 μ g/mL) and LPS of degree are (eventually Concentration be 1 μ g/mL) function cells for 24 hours after, ERK (Figure 16), JNK (Figure 17), P38 (Figure 18) and the P65 of Intracellular phosphorylation (Figure 19) protein expression significantly raises (P < 0.01), to activate MAPK and NF- κ B signal access.

The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention, It should be equivalent substitute mode, be included within the scope of the present invention.

SEQUENCE LISTING

<110>South China Science & Engineering University

<120>a kind of calyx polysaccharide and the preparation method and application thereof from generation to generation

<130> 1

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223>forward of GAPDH

<400> 1

tttgtcaagc tcatttcctg gtatg 25

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223>reverse of GAPDH

<400> 2

tgggataggg cctctcttgc 20

<210> 3

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223>forward of IL-1 β

<400> 3

tgaagggctg cttccaaacc tttgacc 27

<210> 4

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223>reverse of IL-1 β

<400> 4

tgtccattga ggtggagagc tttcagc 27

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223>forward of IL-6

<400> 5

tactcggcaa acctagtgcg 20

<210> 6

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223>reverse of IL-6

<400> 6

gtgtcccaac attcatattg tcagt 25

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223>forward of iNOS

<400> 7

cggcaaacat gacttcaggc 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223>reverse of iNOS

<400> 8

gcacatcaaa gcggccatag 20

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223>forward of TNF-α

<400> 9

ggggattatg gctcagggtc 20

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223>reverse of TNF-α

<400> 10

cgaggctcca gtgaattcgg 20

Claims

1. a kind of calyx Thick many candies from generation to generation, it is characterised in that: the Thick many candies of calyx from generation to generation are prepared by the following preparation method:

(1) will calyx grinding and sieving from generation to generation, obtain calyx powder from generation to generation；Distilled water heating and refluxing extraction calyx powder from generation to generation, then Extracting solution is centrifuged, and supernatant is concentrated under reduced pressure, the Thick many candies solution of calyx from generation to generation being concentrated；The hot water return extracts Time be 2~4 h；

(2) it is mixed by polysaccharide solution and by pretreated macroporous absorbent resin, decolorization filters out polysaccharide solution, is used in combination Water washes out the polysaccharide adsorbed in resin repeatedly, and the polysaccharide solution after decoloration is merged, and is concentrated under reduced pressure, and obtains concentration liquid glucose；Step (2) condition of the decolorization is in 40~60 DEG C of 2~4 h of water-bath；

(3) precipitating reagent is added in concentration liquid glucose to be precipitated, is then centrifuged for, discard supernatant liquid, collect sediment and drying, obtain To calyx Thick many candies from generation to generation；

It is D354FD resin that macroporous absorbent resin described in step (2), which is selected,；Precipitating reagent described in step (3) is dehydrated alcohol Or the ethanol solution that volume fraction is 90~100%；The temperature of the precipitating is 0~4 DEG C.

2. calyx Thick many candies from generation to generation according to claim 1, it is characterised in that:

The temperature being heated to reflux described in step (1) is 75~100 DEG C；The number that the hot water return extracts is 2~4 times.

3. calyx Thick many candies from generation to generation according to claim 1, it is characterised in that:

The pretreated method of macroporous absorbent resin described in step (2) are as follows: macroporous absorbent resin is first used to distilled water immersion 12 Then~24 h first impregnate 0.5~3 h with the hydrochloric acid solution that mass fraction is 3~5%, distillation is washed to neutrality, then uses quality The sodium hydroxide solution that score is 3~5% impregnates 0.5~3 h, and distillation is washed to neutrality, obtains by pretreated macroporous absorption Resin.

4. calyx Thick many candies from generation to generation according to claim 1, it is characterised in that: the additional amount of precipitating reagent described in step (3) For 3~5 times that liquid glucose volume is concentrated；The time of precipitating described in step (3) is 8~24 h；

Ratio of water to material, that is, mass ratio that hot water return as described in step (1) extracts is 15:1~30:1；

5. calyx Thick many candies from generation to generation according to claim 1, it is characterised in that:

The revolving speed of centrifugation described in step (1) is 3000~5000 r/min；The time of the centrifugation is 8~12 min；It is described The number of filtering is 2 ~ 4 times；

The revolving speed of centrifugation described in step (3) is 3000~5000 r/min；The time of the centrifugation is 12~15 min；Step (3) dry temperature described in is 40~60 DEG C.

6. calyx refined polysaccharide from generation to generation, it is characterised in that: by by the described in any item calyx Thick many candies from generation to generation of claim 1 ~ 5 It is further purified to obtain；The refined polysaccharide of calyx from generation to generation is six kinds of calyx refined polysaccharides from generation to generation,

It is homogeneous components that the refined polysaccharide of calyx from generation to generation, which is denoted as CP-I, and Mn molecular weight is 13169 Da；

It is homogeneous components that the refined polysaccharide of calyx from generation to generation, which is denoted as CP-II, and Mn molecular weight is 7538 Da；

The refined polysaccharide of calyx from generation to generation is denoted as the non-homogeneous components of CP-III, CP-IV, CP-V and CP-VI respectively；

The calyx refined polysaccharide from generation to generation, is made by the steps to obtain:

(1) it will be transferred in Z-type chromatographic column, use after 40~60 DEG C of rotary evaporation bubble removings by pretreated DEAE-52 resin Constant flow pump pump distilled water so that filler dress column it is uniform, 5~10 s/ drop of flow velocity after balance；

(2) polysaccharide solution will be configured to by calyx Thick many candies from generation to generation, be transferred in chromatographic column, and successively use distilled water, 0.05 mol/L NaCl, 0.1 mol/L NaCl, 0.15 mol/L NaCl, 0.2 mol/L NaCl, the elution of 0.3 mol/L NaCl solution, stream Fast 5~10 s/ drops, collect each fraction respectively, and phend-sulphuric acid tracing detection eluent measures the absorbance under 490 nm, draws Elution curve processed, the eluent in same absorption peak merge；Then concentrated by rotary evaporation, vacuum freeze drying, by DEAE-52 ion Six components are obtained after exchange column chromatography, wherein the refined polysaccharide that distilled water affords is named as CP-I, 0.05 mol/L The refined polysaccharide that NaCl is afforded is named as CP-II, and the refined polysaccharide that 0.1 mol/L NaCl is afforded is named as CP- III, the refined polysaccharide that 0.15 mol/L NaCl is afforded are named as CP-IV, the purification that 0.2 mol/L NaCl is afforded Polysaccharide is named as CP-V, and the refined polysaccharide that 0.3 mol/L NaCl is afforded is named as CP-VI.

7. calyx refined polysaccharide from generation to generation according to claim 6, it is characterised in that:

The pretreated method of DEAE-52 resin described in step (1) are as follows: DEAE-52 resin is impregnated 12~24 with 4~30 DEG C of water h；0.5~2 h is impregnated with the hydrochloric acid solution of 0.5 mol/L again；The NaOH solution of 0.5 mol/L impregnates 0.5~2 h, filtering, water It is washed till neutrality, is obtained by pretreated DEAE-52 resin.

8. the described in any item Thick many candies of calyx from generation to generation of claim 1 ~ 5 or the described in any item calyx from generation to generation of claim 6~7 Refined polysaccharide is preparing the application in medicament for immunity enhancement, it is characterised in that: the refined polysaccharide of calyx from generation to generation is to be denoted as CP- The calyx refined polysaccharide from generation to generation of II.