CN104031171B - Polysaccharides from Prunella vulgaris L of high molecular and preparation method thereof and the application in immune drug - Google Patents
Polysaccharides from Prunella vulgaris L of high molecular and preparation method thereof and the application in immune drug Download PDFInfo
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Abstract
Polysaccharides from Prunella vulgaris L of the open high molecular of the present invention and preparation method thereof and the application in immune drug.It is 1:15 ~ 1:35 that Spica Prunellae powder and distilled water are made into solid-to-liquid ratio by the method, under being placed in ultrasonic field, power 100 ~ 300W, frequency 15 ~ 50KHz, time 10 ~ 30min, temperature 25 ~ 40 DEG C, then under material pumps into high-voltage field, pressure 100 ~ 500MPa, temperature 100 ~ 130 DEG C, time 10 ~ 30min; Filter, concentrated, deproteinated, decolouring, alcohol precipitation, lyophilize obtains Crude polysaccharides, obtains Polysaccharides from Prunella vulgaris L through column chromatography purification.It is high that the inventive method has polysaccharide yield, molecular weight is 1000 ~ 4000K dalton, it is high molecular mixed polysaccharide, can the significant stimulation mouse macrophage RAW246.7 secretory immune factor, and to the basic no cytotoxicity of human normal cell, there is antitumour activity simultaneously, the immune drug or healthcare products that have no side effect can be developed.
Description
Technical field
The present invention relates to bio-pharmaceuticals engineering and field of health care food, be specifically related to a kind of preparation method with the Polysaccharides from Prunella vulgaris L of immunostimulatory activity and application of high molecular.
Background technology
Spica Prunellae (PrunellavulgarisL.), European Region claims " self ?heal ", and popular name sheep intestines dish, lantern head, iron look grass, excellent column cap flower, large headdress flower etc. are the dry fruit ear of Labiatae Prunella plant Spica Prunellae.Prunella plant is a kind of perennial plant, is distributed widely in Europe and China, and China is mainly distributed in the ground such as Jiangsu, Zhejiang, Anhui and Hubei, and produce 4 kinds and 3 mutation, wherein a kind is introducing culture.It is cold in nature, pungent, bitter, enter liver, gallbladder channel, has clearing away the liver-fire, swelling and pain relieving, the effect such as clearing heat and detoxicating.Spica Prunellae is a kind of draft of integration of drinking and medicinal herbs, and at folks of china, Spica Prunellae is widely used in the healing etc. for the treatment of throat pain, reducing heating and promotion wound as dietotherapy invigorant; The diseases such as jaundice, hepatitis, diabetes and pulmonary tuberculosis are used for the treatment of as Chinese medicine; In South China as one of composition of formula of herbal tea and the beautiful soup of nutrition.Research shows that Spica Prunellae is rich in many important biologically active substances, mainly comprise polysaccharide, triterpene and glycoside thereof, sterol and glycoside thereof, flavonoid, tonka bean camphor, organic acid and volatilization wet goods, its main pharmacological show hypotensive, reducing blood-fat, antiviral, antibacterial, anti-oxidant, immunostimulation and the aspect such as antitumor.
Natural plant polyose has bioactive macromolecular substance as a class, is studied widely in recent years, and modern medicine study shows, polysaccharide has anti-oxidant, immunomodulatory and the multiple biological activity such as antitumor.At present, patent of invention CN1965894A discloses one and has immunocompetent Prunella vulgaris polysaccharide and its preparation method and application.But do not have both at home and abroad and can be used as the preparation of medicine and the report of use.And the report about Polysaccharides from Prunella vulgaris L extracting method mainly adopts conventional hot water's extraction, this kind of method, and because plant cell wall is survivable, extraction time is longer, and extraction yield is low, causes the energy and the wasting of resources.
Summary of the invention
For above-mentioned situation, technical problem solved by the invention is a kind of preparation method and application of Polysaccharides from Prunella vulgaris L of high molecular, provides a kind of and has no side effect and have clinical medicine or the healthcare products of stronger immunostimulatory activity.
Object of the present invention is achieved through the following technical solutions:
A preparation method for the Polysaccharides from Prunella vulgaris L of high molecular, comprises the following steps:
(1) by after the drying of Spica Prunellae fruit ear, pulverize, sieve, add alcohol heating reflux, decolouring and removing small molecular alcohol soluble substance, filtration treatment obtains residue, residue repeats to add alcohol heating reflux 2 ~ 3 times, dry 24 ~ 48h in the air dry oven of 45 ~ 60 DEG C;
(2) the Spica Prunellae sample of step (1) process gained and distilled water being made into solid-to-liquid ratio is 1:15 ~ 1:35, under being placed in ultrasonic field condition, ultrasonic output rating is 100 ~ 300W, and frequency is 15 ~ 50KHz, treatment time 10 ~ 30min, temperature 25 ~ 40 DEG C; Then pump in high-voltage field by material, pressure is 100 ~ 500MPa, and temperature is 100 ~ 130 DEG C, time 10 ~ 30min, then in 5 ~ 10s, standard atmospheric pressure is arrived in decompression, after carrying out supersound process in material blowback ultrasonic field, carry out autoclaving again, loop cycle is 3 ~ 5 times; Described loop cycle refers to that the first supersound process of material carries out a working cycle of autoclaving again; Centrifugation obtains extracting solution, centrifugal force 4500 ~ 10000g, time 5 ~ 10min, and at 45 ~ 65 DEG C, concentrating under reduced pressure is to 1/5 ~ 1/10 of original volume, obtains polysaccharide concentrated solution;
(3) in polysaccharide concentrated solution, add Sevag reagent, the volume ratio of polysaccharide concentrated solution and Sevag reagent is 3:1 ~ 6:1, vibration 20 ~ 30min, centrifugation liquid glucose, and centrifugal force 4500 ~ 10000g, centrifugation time 5 ~ 10min, repeat 10 ~ 20 times;
(4) concentrated solution after deproteinated in (3) is joined macroporous resin, carry out Static Adsorption, the volume ratio of concentrated solution and macroporous resin is 5:1 ~ 2:1, temperature 35 ~ 50 DEG C, and time 4 ~ 6h, filters to get filtrate;
(5) in (4), dehydrated alcohol is added in filtrate, add while stirring, ethanol contend concentration is made to reach 65 ~ 85%, 12 ~ 24h is left standstill in 0 ~ 5 DEG C of refrigerating chamber, the centrifugal polysaccharide that must precipitate, centrifugal force 4500 ~ 10000g, time 10 ~ 15min, lyophilize obtains Spica Prunellae Crude polysaccharides;
(6) Crude polysaccharides is dissolved in distilled water, compound concentration is the polysaccharide soln of 20 ~ 30mg/mL, be splined on DEAE ?Sepharose post, the ratio of applied sample amount volume and column volume is 1:3 ~ 1:5, wash-out is carried out with distilled water, flow velocity 0.5 ~ 2mL/min, is in charge of collection, often pipe 5 ~ 10mL, Phenol sulfuric acid procedure detects polysaccharide, sugared reacting positive is collected liquid merge, concentrating under reduced pressure at 45 ~ 65 DEG C, lyophilize obtains the Polysaccharides from Prunella vulgaris L of purifying; The described positive collects the collection liquid that liquid is 1/2 maximum light absorption value scope.
For realizing the object of the invention further, preferably, described ethanol contend concentration is 95%; Described sieve into cross 30 ~ 50 mesh sieves.In described step (3), Sevag reagent is the mixed solution of chloroform and propyl carbinol, and its volume ratio is 3:1 ~ 6:1.In described step (4), macroporous resin is macroporous resin D101, polyamide resin or macroporous resin D354FD.Polysaccharide molecule weight range prepared by described step (6) is 1000 ~ 4000K dalton.Lyophilize Shi described in step (5) and (6) ?50 ± 5 DEG C, dry 24 ~ 48h under 0.08 ~ 0.1MPa vacuum tightness, in dried sample, the mass percentage of moisture is lower than 4%.Described to add alcohol heating reflux for controlling Spica Prunellae powder and ethanol mass ratio be 1:3 ~ 1:6, temperature 65 ~ 85 DEG C, return time 3 ~ 5h.
A Polysaccharides from Prunella vulgaris L for high molecular, is obtained by above-mentioned preparation method.
The application of described Polysaccharides from Prunella vulgaris L in the clinical medicine and healthcare products of preparation raising immunizing power.
Polysaccharides from Prunella vulgaris L of the present invention has stronger immunostimulatory activity, and to human normal cell's no cytotoxicity, can be used as the clinical medicine or healthcare products that improve immunizing power.Polysaccharides from Prunella vulgaris L of the present invention uses with the oral form with drug administration by injection, mainly makes oral liquid, capsule and injection.
Above operation steps if no special instructions, all by this area routine operation.
Compared with prior art, tool of the present invention has the following advantages and technique effect:
(1) compare conventional hot water's microwave leaching technology, the present invention adopts ultrasonic and cycle of higher pressure treatment technology to carry out Polyose extraction, and extraction time is short, and extraction yield is high, and Polysaccharides from Prunella vulgaris L extraction yield improves 20 ~ 45% after measured, and polysaccharide yield is 7 ~ 10%.
(2) Polysaccharides from Prunella vulgaris L of the present invention, molecular weight ranges is 1000 ~ 4000K dalton, form primarily of pectinose, wood sugar, glucose and semi-lactosi, molar percentage content range is respectively 20 ~ 25%, 50 ~ 60%, 5 ~ 9% and 6 ~ 12%, mainly with 10 ~ 25% 1 → and 1 → 6 glycosidic link, 1 → 3 glycosidic link of 60 ~ 70% connects to form, and the novel mixed polysaccharide of the high molecular of the longer side chain of existence and more branch.
(3) high-molecular-weight dextran of Polysaccharides from Prunella vulgaris L of the present invention owing to being connected with 1 → 6 glycosidic link containing more 1 → 3, there is more branched structure simultaneously, so anti-inflammatory factors can be secreted by significant stimulation mouse macrophage RAW246.5, comprise nitrogen protoxide NO, tumour necrosis factor TNF ?α and interleukin-IL ?6, and to mouse macrophage RAW246.5 no cytotoxicity, there is antitumous effect simultaneously, can be used as non-specific immunity clinical medicine or healthcare products.
Accompanying drawing explanation
Fig. 1 is the uv scan graphic representation of embodiment 1 product
Fig. 2 is the GPC spectrogram of embodiment 1 product
Fig. 3 is the infrared spectrogram of embodiment 1 product
Fig. 4 is that embodiment 1 product secretes the impact of nitrogen protoxide (NO) to mouse macrophage RAW246.5.
Fig. 5 is embodiment 1 product on the impact of mouse macrophage RAW246.5 secreting tumor necrosis factor (TNF ?α).
Fig. 6 is embodiment 1 product on the impact of mouse macrophage RAW246.5 secreting leukocytes mesonium (IL ?6).
Embodiment
The present invention will be further described in conjunction with the embodiments, and embodiments of the present invention and form are not limited thereto.
Embodiment 1
(1) by after the drying of Spica Prunellae fruit ear, pulverize with pulverizer and obtain Spica Prunellae powder, cross 30 mesh sieves, take 300g, add 95% (v/v) ethanol of 900g, 65 DEG C of backflow 3h, filter to obtain residue, repeat aforesaid operations 2 times, residue is dry 24h in the air dry oven of 45 DEG C;
(2) take the Spica Prunellae powder 200g after process in (1), add distilled water and carry out poach lixiviate, feed liquid mass ratio is 1:15, and under being placed in ultrasonic field condition, ultrasonic output rating is 100W, and frequency is 15KHz, process 10min, temperature 25 DEG C; Then material is pumped in high-voltage field, pressure is 100MPa, 10min at 100 DEG C, and then in 5s, standard atmospheric pressure is arrived in decompression, after carrying out supersound process in material blowback ultrasonic field, carry out autoclaving again, loop cycle is 3 times, centrifugal treating extraction fluid, centrifugal force 4500g, time 5min, at 45 DEG C, concentrating under reduced pressure is to 1/5 of original volume, obtains polysaccharide concentrated solution;
(3) in (2) concentrated solution, add Sevag reagent, make the volume ratio of concentrated solution and Sevag reagent be 3:1, vibration 20min, centrifugation liquid glucose, centrifugal force 5500g, time 5min, repetitive operation 10 times;
(4) in (3), add macroporous resin D101 in treatment solution, carry out static state and absorb, make the volume ratio of concentrated solution and macroporous resin be 5:1, temperature 35 DEG C, time 4h, filters to get filtrate;
(5) in (4) filtrate, dehydrated alcohol is added, add while stirring, ethanol contend concentration is made to reach 65%, 12h is left standstill at 4 DEG C, the polysaccharide that centrifugal 10min must precipitate, centrifugal force 4500g, lyophilize 36h, obtain Spica Prunellae Water-soluble polysccharide, the extraction yield obtaining Spica Prunellae Crude polysaccharides is after testing 7.53%.
(6) purifying: Crude polysaccharides is dissolved in distilled water, compound concentration is the polysaccharide soln of 20mg/mL, be splined on DEAE ?Sepharose post, the ratio of applied sample amount volume and column volume is 1:3, carries out wash-out, flow velocity 1mL/min with distilled water solution, be in charge of collection, often pipe 5mL, Phenol sulfuric acid procedure detects polysaccharide, sugared reacting positive is collected liquid and merges, concentrating under reduced pressure at 45 DEG C, lyophilize 36h obtains the Polysaccharides from Prunella vulgaris L of purifying, its productive rate 3.12%, cotton-shaped in white cotton.
The qualification of Polysaccharides from Prunella vulgaris L:
(1) measurement of the polysaccharide content in sample: polysaccharide content %=total sugar content/sample quality × 100.Total sugar content adopt Ben Fen ?sulfuric acid process measure.In this Polysaccharides from Prunella vulgaris L, sugared content is 98.5% after measured.
(2) solvability measures: take 10mg Polysaccharides from Prunella vulgaris L, be placed in 10mL centrifuge tube, add 5mL distilled water, vortex oscillation 10min, under centrifugal force 10000g, centrifugal 10 minutes, observes bottom centrifuge tube with or without precipitation.Observe and find that Polysaccharides from Prunella vulgaris L solution is through centrifugal nothing precipitation, shows that the water-soluble of this Polysaccharides from Prunella vulgaris L is better.
(3) Dian ?potassiumiodide reaction experiment: compound concentration is the Polysaccharides from Prunella vulgaris L solution of 1mg/mL, adds the iodine reagent of 1.2mL (containing the I of 0.02%
2with 0.2% KI solution), after mixing 300 ?carry out UV scanning in 700nm wavelength region.Result shows that the reaction solution of polysaccharide sample and iodine reagent has maximum absorption band at 360nm place, and at 565nm place without maximum absorption, proves that this Polysaccharides from Prunella vulgaris L exists longer side chain and more branch.
(4) uv scan: preparation polysaccharide concentration is the solution of 1mg/mL take distilled water as contrast, then 190 ?carry out UV scanning in 400nm wavelength region.As shown in Figure 1, this polysaccharide sample does not have uv-absorbing at 260nm, 280nm wavelength place, proves this Polysaccharides from Prunella vulgaris L not containing protein and nucleic acid.
(5) mensuration of molecular weight: the molecular weight HPPC of polysaccharide sample measures, adopts the gel permeation chromatograph (WaterBreezeGPC) of U.S. Waters Ltd., chromatographic column be TSK ?G5000PW
xLwith TSK ?G3000PW
xLcolumns in series, moving phase is 0.02moL/L phosphate buffer solution (PH6.0); Flow velocity 0.6mL/min; Column temperature 35 DEG C, detector is 2414 type differential refraction detectors.As shown in accompanying drawing 2GPC collection of illustrative plates, calculating Polysaccharides from Prunella vulgaris L according to typical curve is that molecular-weight average is about 1700K dalton.
(6) Infrared spectroscopy: get polysaccharide sample and be about 2mg, by with KBr mixed pressuring plate, put into Fourier transform infrared spectroscopy, 400 ?4000cm
?1interval is scanned, the infrared absorption pattern of collected specimens.As shown in accompanying drawing 3 infrared spectra (IR), polysaccharide sample is at 3422cm
?1near broad peak be O ?H key stretching vibration produce, 2925cm
?1the more weak peak of left and right be C ?the stretching vibration of H key, at 1422cm
?1near all have very little absorption peak be C ?the angle vibration peak of H, prove that this Polysaccharides from Prunella vulgaris L is a kind of polysaccharose substance.
(7) monose composition measuring: polysaccharide sample first through being hydrolyzed into monose, then generates sugared nitrile acetic ester derivative after derivatize, adopts the monose composition in gas chromatographic analysis polysaccharide sample, gas chromatographic column be TR ?5MS elastic capillary-column, carrier gas is 99.999% high-purity helium, heating schedule: initial column temperature 100 DEG C, keeps 2min, 280 DEG C are risen to the heat-up rate of 5 DEG C/min, keep 5min, flow velocity 1mL/min, sample size 1 μ L, splitting ratio 10:1, injector temperature 250 DEG C.Result shows that this Polysaccharides from Prunella vulgaris L sample is containing pectinose, wood sugar, seminose, glucose and semi-lactosi composition, and mole percent level is respectively 24.2%, 55.9%, 1.9%, 8.3%, 9.7%.
(8) periodate oxidation and Smith degraded: polysaccharide sample through periodate oxidation, and then after carrying out Smith degraded, carries out derivatization treatment and prepares sugared nitrile acetonyl ester derivative.Adopt gas chromatograph Agilent6890T to detect, gas chromatographic column HP ?1701 capillary columns, detector temperature 270 DEG C; Temperature of vaporization chamber 220 DEG C, heating schedule: initial temperature 180 DEG C, rises to 220 DEG C with the heat-up rate of 2 DEG C/min, keeps 1min, rises to 260 DEG C with the heat-up rate of 5 DEG C/min; Carrier gas gas velocity 40mL/min, air velocity is 450mL/min, and nitrogen flow rate is 25mL/min, does not shunt.Result shows that Polysaccharides from Prunella vulgaris L contains 1 → and 1 → 6 glycosidic link, 1 → 2 and 1 → 4 glycosidic link of 13.1% and 1 → 3 glycosidic link of 63.4% of 23.5%.
In sum, the polysaccharide of the present embodiment is a kind of cotton-shaped in white cotton, polysaccharide content reaches more than 98%, good water solubility, not containing the impurity such as protein and nucleic acid, form containing pectinose, wood sugar, seminose, glucose and semi-lactosi, mole percent level is respectively 24.2%, 55.9%, 1.9%, 8.3%, 9.7%, by 1 → and 1 → 6 glycosidic link of 23.5%, 13.1% 1 → 2 to be connected with 1 → 3 glycosidic link of 1 → 4 glycosidic link and 63.4%, there is the novel mixed polysaccharide of the high molecular of longer side chain and more branch in glycosidic link.
Embodiment 2
(1) by after the drying of Spica Prunellae fruit ear, pulverize with pulverizer and obtain Spica Prunellae powder, cross 50 mesh sieves, take 300g, add 95% (v/v) ethanol of 1800g, 85 DEG C of backflow 5h, filter to obtain residue, repeat aforesaid operations 3 times, residue is dry 48h in the air dry oven of 60 DEG C;
(2) take the Spica Prunellae powder 200g after process in (1), add distilled water and carry out poach lixiviate, feed liquid mass ratio is 1:35, and under being placed in ultrasonic field condition, ultrasonic output rating is 300W, and frequency is 50KHz, process 30min, temperature 40 DEG C; Then material is pumped in high-voltage field, pressure is 500MPa, 30min at 130 DEG C, and then in 8s, standard atmospheric pressure is arrived in decompression, after carrying out supersound process in material blowback ultrasonic field, carry out autoclaving again, loop cycle is 5 times, centrifugal treating extraction fluid, centrifugal force 10000g, time 10min, at 65 DEG C, concentrating under reduced pressure is to 1/10 of original volume, obtains polysaccharide concentrated solution;
(3) in (2) concentrated solution, add Sevag reagent, make the volume ratio of concentrated solution and Sevag reagent be 6:1, vibration 30min, centrifugation liquid glucose, centrifugal force 10000g, time 10min, repetitive operation 20 times;
(4) in (3), add macroporous resin D101 in treatment solution, carry out static state and absorb, make the volume ratio of concentrated solution and macroporous resin be 2:1, temperature 50 C, time 6h, filters to get filtrate;
(5) in (4) filtrate, dehydrated alcohol is added, add while stirring, ethanol contend concentration is made to reach 85%, 24h is left standstill at 4 DEG C, the polysaccharide that centrifugal 15min must precipitate, centrifugal force 10000g, lyophilize 48h, obtain Spica Prunellae Water-soluble polysccharide, obtain the extraction yield 10.13% of Spica Prunellae Crude polysaccharides after testing.
(6) purifying: Crude polysaccharides is dissolved in distilled water, compound concentration is the polysaccharide soln of 30mg/mL, be splined on DEAE ?Sepharose post, the ratio of applied sample amount volume and column volume is 1:5, carries out wash-out, flow velocity 0.5mL/min with distilled water solution, be in charge of collection, often pipe 5mL, Phenol sulfuric acid procedure detects polysaccharide, sugared reacting positive is collected liquid and merges, concentrating under reduced pressure at 65 DEG C, lyophilize 48h obtains the Polysaccharides from Prunella vulgaris L of purifying, its productive rate 4.82%, cotton-shaped in white cotton.Its molecular-weight average is about 1680K dalton, and other result is substantially with embodiment 1.
Embodiment 3
(1) by after the drying of Spica Prunellae fruit ear, pulverize with pulverizer and obtain Spica Prunellae powder, cross 40 mesh sieves, take 300g, add 95% (v/v) ethanol of 1200g, 75 DEG C of backflow 4h, filter to obtain residue, repeat aforesaid operations 2 times, residue is dry 30h in the air dry oven of 55 DEG C;
(2) take the Spica Prunellae powder 200g after process in (1), add distilled water and carry out poach lixiviate, feed liquid mass ratio is 1:20, and under being placed in ultrasonic field condition, ultrasonic output rating is 180W, and frequency is 30KHz, process 20min, temperature 30 DEG C; Then material is pumped in high-voltage field, pressure is 300MPa, 20min at 115 DEG C, and then in 10s, standard atmospheric pressure is arrived in decompression, after carrying out supersound process in material blowback ultrasonic field, carry out autoclaving again, loop cycle is 4 times, centrifugal treating extraction fluid, centrifugal force 8000g, time 7min, at 55 DEG C, concentrating under reduced pressure is to 1/8 of original volume, obtains polysaccharide concentrated solution;
(3) in (2) concentrated solution, add Sevag reagent, make the volume ratio of concentrated solution and Sevag reagent be 4:1, vibration 25min, centrifugation liquid glucose, centrifugal force 6000g, time 8min, repetitive operation 15 times;
(4) in (3), add macroporous resin D101 in extracting solution, carry out static state and absorb, make the volume ratio of concentrated solution and macroporous resin be 3:1, temperature 45 C, time 5h, filters to get filtrate;
(5) in (4) filtrate, dehydrated alcohol is added, add while stirring, ethanol contend concentration is made to reach 75%, 18h is left standstill at 4 DEG C, the polysaccharide that centrifugal 12min must precipitate, centrifugal force 7000g, lyophilize 40h, obtain Spica Prunellae Water-soluble polysccharide, the extraction yield obtaining Spica Prunellae Crude polysaccharides is after testing 8.89%.
(6) Crude polysaccharides is dissolved in distilled water, compound concentration is the polysaccharide soln of 25mg/mL, be splined on DEAE ?Sepharose post, the ratio of applied sample amount volume and column volume is 1:4, carries out wash-out, flow velocity 1.5mL/min with distilled water solution, be in charge of collection, often pipe 5mL, Phenol sulfuric acid procedure detects polysaccharide, sugared reacting positive is collected liquid and merges, 50 DEG C of rotary evaporations concentrate, lyophilize 36h obtains the Polysaccharides from Prunella vulgaris L of purifying, its productive rate 4.05%, cotton-shaped in white cotton.Its molecular-weight average is about 1770K dalton, and other result is substantially with embodiment 1.
Embodiment 4
The immunostimulatory activity experiment of embodiment 1 Polysaccharides from Prunella vulgaris L
Experimental cell: mouse macrophage RAW246.7
Cell cultures: be scattered in fresh culture by mouse macrophage RAW246.7, and be inoculated in culturing bottle, is placed in 37 DEG C, 5% (volume fraction) CO
2quiescent culture in the incubator of concentration.Fresh culture is for containing 10% (by percentage to the quality) foetal calf serum, 100 μ g/mL Streptomycin sulphates, the DMEM substratum of 100 units/mL penicillin.
(1) the mouse macrophage RAW246.7 taken the logarithm vegetative period, adds fresh culture, and piping and druming is prepared into single cell suspension, after blood counting chamber counting, by cell concn adjustment about 1 × 10
6individual/mL, i.e. cell suspending liquid, be inoculated in 96 porocyte culture plates (100 μ L/ hole), is placed in 37 DEG C, 5% CO2gas incubator cultivates.Zeroing group, experimental group, blank group and positive controls are established in experiment.Zeroing group is not containing the blank well of 100 μ L cells, only adds during experiment; Experimental group, blank group and positive controls are the hole containing 100 μ L cell suspending liquids.Dull and stereotyped outermost edge adds aseptic PBS solution, every hole 200 μ L.
(2) after 24h, after cell attachment, discard substratum, 100 μ L fresh cultures are added toward zeroing group and blank group, experimental group adds the polysaccharide sample 100 μ L of the different concns of 62.5,125,250,500,1000 μ g/mL respectively, add the lipopolysaccharides (LPS, 50 μ g/mL) (liquid and positive control are all with not preparing containing blood serum medium) of 100 μ L in positive controls, 5 parallel holes established by each different components sample.
(3) continue to be placed in 37 DEG C, 5% CO2gas incubator cultivates 24h.Use nitrogen protoxide NO in Elisa kit detection cell supernatant liquor respectively, tumour necrosis factor TNF ?α and interleukin-IL ?6 content.Detection method adopts NO testing cassete (A013 ?2) to buy and build up Bioengineering Research Institute from Nanjing; Mouse TNF ?α ELISA kit (EMC102a), mouse IL ?6ELISA test kit (EMC004) buy from Xin Bosheng bio tech ltd.
Experimental result is as accompanying drawing 4,5,6, compared with blank group, the polysaccharide energy significant stimulation mouse macrophage RAW246.7 of different concns secrete anti-inflammatory factors NO, TNF ?α and IL ?6, along with the increase of polysaccharide concentration, NO in cell conditioned medium liquid, TNF ?α and IL ?6 content also increase gradually, when polysaccharide concentration is 1000 μ g/mL, NO in cell conditioned medium liquid, TNF ?α and IL ?6 content be respectively 10 μm of ol/L, 850pg/mL, 1600pg/mL.Result shows that this polysaccharide has significant immunostimulatory activity.The immunostimulatory activity of the polysaccharide sample in other embodiment is similar to embodiment 1 sample.
Embodiment 5
The cytotoxicity Shi Yan of embodiment 1 Polysaccharides from Prunella vulgaris L ?mtt assay
Cell cultures: cultural method is with embodiment 5.
(1) the mouse macrophage RAW246.7 taken the logarithm vegetative period, piping and druming is prepared into single cell suspension, adds fresh culture, after blood counting chamber counting, by cell concn adjustment about 5 × 10
4individual/mL, i.e. cell suspending liquid, be inoculated in 96 porocyte culture plates (100 μ L/ hole), is placed in 37 DEG C, 5% (volume fraction) CO2gas incubator cultivates.Zeroing group, experimental group and control group are established in experiment.Zeroing group does not add cell, adds 100 μ L substratum, experimental group and control group, and every hole adds 100 μ L cell suspending liquids.Dull and stereotyped outermost edge adds aseptic PBS solution, every hole 200 μ L.
(2) after 24h, after cell attachment, discard substratum, 100 μ L fresh mediums are added toward zeroing group and control group, experimental group adds the polysaccharide sample 100 μ L of the different concns of 62.5,125,250,500,1000 μ g/mL respectively, all samples is prepared by substratum, and 5 parallel holes established by each different components sample.
(3) continue to be placed in 37 DEG C, 5% CO2gas incubator cultivates 48h, every Kong Jun adds the MTT solution (5mg/mL) of 10 μ L, continue to cultivate 4h, then substratum is discarded, every hole adds the DMSO of 100 μ L, and vibrator vibrates 10min, measures light absorption value A at microplate reader OD570nm place, and calculate cell survival rate, calculation formula is as follows:
Cell survival rate %=(As ?Ao)/(Ac ?Ao) × 100
Wherein As is the light absorption value of experimental group, and Ac is the light absorption value of control group, and Ao is the light absorption value of zeroing group.
Experimental result is as table 1, when polysaccharide sample concentration is lower than 250 μ g/mL, cell survival rate increases, show that the polysaccharide sample of low dosage can promote that mouse macrophage RAW246.5 breeds, when polysaccharide concentration is increased to 1000 μ g/mL, cell survival rate is greater than 90%, shows that this polysaccharide sample does not have cytotoxicity substantially to mouse macrophage RAW246.7.The cytotoxicity of the polysaccharide sample in other embodiment is similar to embodiment 1 sample.
Table 1
| Dosage (μ g/mL) | Cell survival rate % |
| Blank | 100 |
| 62.5 | 105.37±2.85 |
| 125 | 107.48±1.22 |
| 250 | 101.86±2.07 |
| 500 | 94.69±3.56 |
| 1000 | 91.65±2.16 |
Embodiment 6 Polysaccharides from Prunella vulgaris L oral liquid
Get Polysaccharides from Prunella vulgaris L and be dissolved in aseptic ultrapure water, be made into the solution that concentration is 0.5mg/mL, in Polysaccharides from Prunella vulgaris L quality, add the Sodium Benzoate of 0.15%, the glucose of 5.0%, the Citric Acid of 0.2%, ultraviolet disinfection, canned in Brown Glass Brown glass bottles and jars only, often prop up 5mL, namely obtain.Oral, a 5mL, each 1 time sooner or later.Function with cure mainly: immunomodulator, for the assisting therapy of the immunologic function and malignant tumour that improve human organism, is applicable to any crowd.
Embodiment 7 Polysaccharides from Prunella vulgaris L capsule
Get Polysaccharides from Prunella vulgaris L and be dissolved in aseptic ultrapure water, add the resistant dextrin of 1000 times of Polysaccharides from Prunella vulgaris L quality, starch and Sodium Benzoate, by percentage to the quality, wherein resistant dextrin content is 50.0%, and starch content is 49.5%, and Sodium Benzoate content is 0.5%, abundant mixing, the concentration regulating mixture is 25%, carries out spraying dry (inlet temperature 160 DEG C, air outlet temperature 90 DEG C), after sterilizing, incapsulate in shell, every ball 0.4g, oral, 2 balls, 1 time on the one.Function with cure mainly: immunomodulator, for the assisting therapy of the immunologic function and malignant tumour that improve human organism, is applicable to any crowd.
Embodiment 8 Polysaccharides from Prunella vulgaris L injection liquid
Polysaccharides from Prunella vulgaris L is dissolved in aseptic ultrapure water, is made into the solution that concentration is 0.5mg/mL, ultraviolet disinfection, be divided in the brown apothecary jar of 5mL, every bottle of 2mL.Add 250mL physiological saline during use or 5% glucose injection dilutes, adopt intravenous drip to use.Function with cure mainly: immunomodulator, for the assisting therapy of the immunologic function and malignant tumour that improve human organism, is applicable to any crowd.
Claims (9)
1. a preparation method for the Polysaccharides from Prunella vulgaris L of high molecular, is characterized in that comprising the following steps:
(1) by after the drying of Spica Prunellae fruit ear, pulverize, sieve, add alcohol heating reflux, decolouring and removing small molecular alcohol soluble substance, filtration treatment obtains residue, residue repeats to add alcohol heating reflux 2 ~ 3 times, dry 24 ~ 48h in the air dry oven of 45 ~ 60 DEG C;
(2) the Spica Prunellae sample of step (1) process gained and distilled water being made into solid-to-liquid ratio is 1:15 ~ 1:35, under being placed in ultrasonic field condition, ultrasonic output rating is 100 ~ 300W, and frequency is 15 ~ 50KHz, treatment time 10 ~ 30min, temperature 25 ~ 40 DEG C; Then pump in high-voltage field by material, pressure is 100 ~ 500MPa, and temperature is 100 ~ 130 DEG C, time 10 ~ 30min, then in 5 ~ 10s, standard atmospheric pressure is arrived in decompression, after carrying out supersound process in material blowback ultrasonic field, carry out autoclaving again, loop cycle is 3 ~ 5 times; Described loop cycle refers to that the first supersound process of material carries out a working cycle of autoclaving again; Centrifugation obtains extracting solution, centrifugal force 4500 ~ 10000g, time 5 ~ 10min, and at 45 ~ 65 DEG C, concentrating under reduced pressure is to 1/5 ~ 1/10 of original volume, obtains polysaccharide concentrated solution;
(3) in polysaccharide concentrated solution, add Sevag reagent, the volume ratio of polysaccharide concentrated solution and Sevag reagent is 3:1 ~ 6:1, vibration 20 ~ 30min, centrifugation liquid glucose, and centrifugal force 4500 ~ 10000g, centrifugation time 5 ~ 10min, repeat 10 ~ 20 times;
(4) the polysaccharide concentrated solution after deproteinated in (3) is joined macroporous resin, carry out Static Adsorption, the volume ratio of concentrated solution and macroporous resin is 5:1 ~ 2:1, temperature 35 ~ 50 DEG C, and time 4 ~ 6h, filters to get filtrate;
(5) in (4), dehydrated alcohol is added in filtrate, add while stirring, ethanol contend concentration is made to reach 65 ~ 85%, 12 ~ 24h is left standstill in 4 ~ 5 DEG C of refrigerating chambers, the centrifugal polysaccharide that must precipitate, centrifugal force 4500 ~ 10000g, time 10 ~ 15min, lyophilize obtains Spica Prunellae Crude polysaccharides;
(6) Spica Prunellae Crude polysaccharides is dissolved in distilled water, compound concentration is the polysaccharide soln of 20 ~ 30mg/mL, be splined on DEAE ?Sepharose post, the ratio of applied sample amount volume and column volume is 1:3 ~ 1:5, wash-out is carried out with distilled water, flow velocity 0.5 ~ 2mL/min, is in charge of collection, often pipe 5 ~ 10mL, Phenol sulfuric acid procedure detects polysaccharide, sugared reacting positive is collected liquid merge, concentrating under reduced pressure at 45 ~ 65 DEG C, lyophilize obtains the Polysaccharides from Prunella vulgaris L of purifying; The described positive collects the collection liquid that liquid is 1/2 maximum light absorption value scope.
2. the preparation method of the Polysaccharides from Prunella vulgaris L of a kind of high molecular according to claim 1, is characterized in that, described ethanol contend concentration is 95%; Described sieve into cross 30 ~ 50 mesh sieves.
3. the preparation method of the Polysaccharides from Prunella vulgaris L of a kind of high molecular according to claim 1, is characterized in that, in described step (3), Sevag reagent is the mixed solution of chloroform and propyl carbinol, and its volume ratio is 3:1 ~ 6:1.
4. the preparation method of the Polysaccharides from Prunella vulgaris L of a kind of high molecular according to claim 1, is characterized in that, in described step (4), macroporous resin is macroporous resin D101, polyamide resin or macroporous resin D354FD.
5. the preparation method of the Polysaccharides from Prunella vulgaris L of a kind of high molecular according to claim 1, is characterized in that, polysaccharide molecule weight range prepared by described step (6) is 1000 ~ 4000K dalton.
6. the preparation method of the Polysaccharides from Prunella vulgaris L of a kind of high molecular according to claim 1, it is characterized in that, lyophilize Shi described in step (5) and (6) ?50 ± 5 DEG C, dry 24 ~ 48h under 0.08 ~ 0.1MPa vacuum tightness, in dried sample, the mass percentage of moisture is lower than 4%.
7. the preparation method of the Polysaccharides from Prunella vulgaris L of a kind of high molecular according to claim 1, is characterized in that, described in add alcohol heating reflux for controlling Spica Prunellae powder and ethanol mass ratio be 1:3 ~ 1:6, temperature 65 ~ 85 DEG C, return time 3 ~ 5h.
8. a Polysaccharides from Prunella vulgaris L for high molecular, is characterized in that it is obtained by preparation method described in any one of claim 1 ~ 7.
9. the application of Polysaccharides from Prunella vulgaris L in the clinical medicine and healthcare products of preparation raising immunizing power according to claim 8.
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| CN111965271A (en) * | 2020-07-17 | 2020-11-20 | 佛山科学技术学院 | Quantitative method for polysaccharide absorption |
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| CN1965894A (en) * | 2006-05-12 | 2007-05-23 | 上海中医药大学 | Prunella vulgaris polysaccharide having immune activity, preparation method and application thereof |
| CN102827299A (en) * | 2012-08-27 | 2012-12-19 | 福建农林大学 | Efficient extraction and purification method for kumquat polysaccharide |
| CN103626880A (en) * | 2012-08-24 | 2014-03-12 | 复旦大学 | Prunella vulgaris polysaccharide, and preparation method and purpose thereof |
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| CN1965894A (en) * | 2006-05-12 | 2007-05-23 | 上海中医药大学 | Prunella vulgaris polysaccharide having immune activity, preparation method and application thereof |
| CN103626880A (en) * | 2012-08-24 | 2014-03-12 | 复旦大学 | Prunella vulgaris polysaccharide, and preparation method and purpose thereof |
| CN102827299A (en) * | 2012-08-27 | 2012-12-19 | 福建农林大学 | Efficient extraction and purification method for kumquat polysaccharide |
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