CN107286265B - It is a kind of neutrality notoginseng polysaccharide method for extraction and purification and promote cell Proliferation pharmacology activity research with application - Google Patents
It is a kind of neutrality notoginseng polysaccharide method for extraction and purification and promote cell Proliferation pharmacology activity research with application Download PDFInfo
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Abstract
The present invention relates to a kind of method for extraction and purification of neutral notoginseng polysaccharide and promote cell Proliferation pharmacology activity research and application, using the Radix Notoginseng waste residue for the notoginsenoside extracted in production as raw material, using water extraction and alcohol precipitation method, obtain Radix Notoginseng Thick many candies, Radix Notoginseng Thick many candies are purified using DEAE Sepharose Fast Flow anion-exchange chromatographies, obtain 5 components：Neutral notoginseng polysaccharide PNPS I and 5 acid notoginseng polysaccharide PNPS II, PNPS III, PNPS IV, PNPS V samples, study its influence to Periodontal ligament stem cell and mouse bone-forming cell and application on human skin epidermal cell in-vitro multiplication, prove that neutrality notoginseng polysaccharide PNPS I promote periodontal ligament stem cell and mouse bone-forming cell and the effect of application on human skin epidermal cell proliferation most apparent, neutral notoginseng polysaccharide PNPS I can be used as the important drugs intermediate of developing new drug, the natural material of functional health-care food, and it is alternatively arranged as the activated feedstock of daily chemical products.
Description
Technical field
The invention belongs to field of medicaments, are related to a kind of method for extraction and purification and promotion cell Proliferation medicine of neutral notoginseng polysaccharide
Manage activity research and application.
Background technology
Polysaccharide is widely present in animal, plant and microorganism.One of constituent as living matter, it joins extensively
With the various biological phenomenas of cell and the adjusting of physiology course, such as the transmission and impression of information between immunocyte, cell turns
The activities such as change, division and regeneration.Since the 1980s, scientists produce the research of herbal polysaccharide dense emerging
Interest.Up to the present the polysaccharide reported has more than 100 kinds, including panaxan, lentinan, ganoderma lucidum polysaccharide, astragalus polyose, Poria cocos
Polysaccharide, Cordyceps sinensis polysaccharide, tremella polysaccharides, Radix Et Caulis Acanthopanacis Senticosi polysaccharide etc. have antiviral, anti-infective, antitumor, anti-oxidant, radioresistance, drop
Blood glucose, liver protection adjust the physiological activity such as immune, and Small side effects
Radix Notoginseng is Araliaceae Panax herbaceos perennial also known as radix notoginseng, pseudo-ginseng etc., and the genuine place of production is Yunnan text
Mountain.Radix Notoginseng is China's valuable ingredients of traditional Chinese medicine, and active ingredient includes saponin(e, dencichine, flavones, polysaccharide, carbene alcohol, amino acid, fat
Fat acid and cyclic peptide etc..Saponin constituent is concentrated mainly on to the research and utilization of compound components of panax notoginseng at present, after saponin constituent extraction,
Other active ingredients are taken as waste residue and liquid to be discarded.
The utilization and use of saponin active ingredient are concentrated mainly on to the research of Radix Notoginseng at present, to other active ingredients
Research is insufficient.The long-term cultivated area of Radix Notoginseng of the genuine place of production Yunnan mountain of papers of Radix Notoginseng has reached 120,000 mu or so, 9000 tons of yield.Often
Year for extract the Radix Notoginseng of arasaponin to be about 1400 tons, studies have shown that after saponin extraction, notoginseng polysaccharide in pseudo-ginseng slag,
It is about 2% to measure content of the notoginseng polysaccharide in Radix Notoginseng, discards nearly 30 tons of notoginseng polysaccharide every year.At present to the research of notoginseng polysaccharide
Report is concentrated mainly on raising studies on immune function.Therefore, classification development can give full play to the product right and wrong of notoginseng polysaccharide effect
It is often necessary.
The patent of invention that number of patent application is 201210058942.6 discloses a kind of notoginseng polysaccharide with physiological activity
Preparation method and applications.This method application resin column chromatography, activated carbon and membrane separation technique purifying and etc., polishing purification
Obtain notoginseng polysaccharide.Have protein, heavy metal and ash content low with the notoginseng polysaccharide that the preparation method of the invention obtains, quality is steady
Fixed feature, and with adjusting immune, protection hepatic injury, reducing the physiological activity such as blood glucose, antitumor, it can conduct alone or in combination
The raw material of natural drug and health-oriented products, but the invention is not purified, is also being promoted carefully without disclosing notoginseng polysaccharide
Born of the same parents are proliferated the effect in terms of pharmacological activity.
The patent of invention that number of patent application is 201510407936.0 discloses a kind of Radix et Rhizoma Gynurae divaricatae polysaccharide and its is preparing
For the application in immunological regulation and anti-tumor drug and functional food, Radix et Rhizoma Gynurae divaricatae polysaccharide can promote list made from the invention
Core macrophage strain swallows dimethyl diaminophenazine chloride and release nitric oxide；It can promote the proliferation of B cell and the proliferation of T cell；It can also be bright
It is aobvious to improve immunosuppressed mice spleen and thymus index, raising in the proliferation of T cell and B cell, improve immunosuppressed mice
Leucocyte level；The growth of tumor tissues can obviously be inhibited；But also body can be protected, inhibit the decline of weight, and increase
Weight；The migration of venous endothelial cell can obviously be inhibited.And polysaccharide is the main composition of Radix et Rhizoma Gynurae divaricatae, has a variety of guarantors
Strong effect, source belongs to pure natural, without side-effects, can be developed into immunological regulation, antitumor, anti-angiogenesis drug and health care
Product, but the patent is concentrated mainly in terms of concrete application there is no being purified with regard to notoginseng polysaccharide and improves immune function, resists and swell
Tumor etc..
The patent of invention that number of patent application is 201210481377.4 discloses a kind of notoginseng polysaccharide extract and its preparation
Method and preparation and application, the notoginseng polysaccharide extract be using extract the waste residue after arasaponin it is extracted as raw material,
Concentration, refined, dry, crushing, checking procedure obtain, and wherein notoginseng polysaccharide content is not less than 15%.The notoginseng polysaccharide carries
It takes the preparation method of object to include extraction, concentration, refined, drying, crush, checking procedure, specifically includes：Arasaponin will be extracted
Purified water extraction is added in waste residue afterwards, filters, and ethanol precipitation is added in concentration, filters, and refines, is dried to obtain object dry product；
The notoginseng polysaccharide extract is added pharmaceutic adjuvant and powder, capsule, granule, tablet or pill is made；The Radix Notoginseng
Polyoses extract is used to prepare the application in antitumor drug and strengthen immunity drug.But the invention is only preliminary extraction,
Do not carry out chromatographic purifying, in terms of concrete application also without reference to.
Invention content
For overcome the deficiencies in the prior art, the present invention provides method for extraction and purification and the promotion of a kind of neutral notoginseng polysaccharide
Cell Proliferation pharmacology activity research and application, to extract the Radix Notoginseng waste residue of notoginsenoside in production as raw material, using water extracting alcohol
Heavy method, obtains Radix Notoginseng Thick many candies, and Radix Notoginseng Thick many candies are purified using DEAE Sepharose Fast Flow anion-exchange chromatographies,
Through dialysis purification, neutral notoginseng polysaccharide PNPS I and acid notoginseng polysaccharide PNPS II, acid notoginseng polysaccharide are obtained after freeze-drying
PNPS III, acid notoginseng polysaccharide PNPS IV, acidity notoginseng polysaccharide PNPS 5 samples of V.Measure Radix Notoginseng Thick many candies, neutrality three
Seven polysaccharide PNPS I and acid notoginseng polysaccharide II-V (acid notoginseng polysaccharide PNPS II, acid notoginseng polysaccharide PNPS III, acidity
Notoginseng polysaccharide PNPS IV, acidity notoginseng polysaccharide PNPS V) content and molecular weight ranges, study it to Periodontal ligament stem cell
With the influence of mouse bone-forming cell and application on human skin epidermal cell in-vitro multiplication, it was demonstrated that neutral notoginseng polysaccharide PNPS I promote periodontal
Film stem cell and mouse bone-forming cell and the effect of application on human skin epidermal cell proliferation are most apparent, and neutral notoginseng polysaccharide can PNPS I works
For the important drugs intermediate of developing new drug, the natural material of functional health-care food, and it is alternatively arranged as the work of daily chemical products
Property raw material.
The present invention provides the following technical solutions：
A kind of method for extraction and purification of neutrality notoginseng polysaccharide, uses the Radix Notoginseng waste residue for extracting notoginsenoside for raw material, wraps
Include following steps：
(1) water extraction and alcohol precipitation method extracts Radix Notoginseng Thick many candies；
(2) DEAE Sepharose Fast Flow anion-exchange chromatographies and dialysis purify Thick many candies, freeze-drying
It is more that method obtains neutral notoginseng polysaccharide PNPS I and acid notoginseng polysaccharide PNPS II, acid notoginseng polysaccharide PNPS III, acid Radix Notoginseng
Sugared PNPS IV, acid notoginseng polysaccharide PNPS V freeze-dried powders.
Preferably, water extraction and alcohol precipitation method extraction Radix Notoginseng Thick many candies specific method is in the step (1)：It takes and extracted Radix Notoginseng
Water is added in the waste residue of total saposins, and boiling water bath boils, and collects filtrate, and centrifugation takes supernatant；It after spin concentration, stands, centrifugation, it is heavy to abandon
It forms sediment, is slowly added to absolute ethyl alcohol, it is stirring while adding, after placement, filter to obtain precipitation；Ethyl alcohol washing precipitation, after ethyl alcohol volatilization is done
With appropriate water dissolution, supernatant is collected in centrifugation, and absolute ethyl alcohol is added, and after placement, filters to obtain precipitation, ethyl alcohol washing precipitation, baking oven dries
It does to get Radix Notoginseng Thick many candies.The waste residue of the present invention is provided by Kunming Medicine Group Stock Co., Ltd.
Any of the above-described scheme is preferably, DEAE Sepharose Fast Flow anion exchanges in the step (2)
The method of chromatography and dialysis purifying Thick many candies includes dress column, balance, loading, elution, dialysis and freeze-drying.
Any of the above-described scheme is preferably, and the specific method of the dress column is：Ethyl alcohol is added, filters, homogenate is made into water,
Using wet method dress post, it is poured slowly into column along column wall with glass bar guiding homogenate；Pillar liquid outlet is opened, makes gel in column
Free settling washes with water chromatographic column edge.
Any of the above-described scheme is preferably, and the specific method of the wet method dress post is：Pillar fills in cotton, is added and steams in column
Distilled water simultaneously keeps a bit of liquid level, and glass bar pressing cotton dispels bubble.
Any of the above-described scheme is preferably, and the specific method of the balance is：Distilled water filter membrane, with more times of amount volume of water
To rinse pillar, until efflux conductivity and pH are constant, balance 48 hours.
Any of the above-described scheme is preferably, and the specific method of the loading is：Radix Notoginseng Thick many candies are dissolved in a small amount of distilled water,
Centrifugation, supernatant liquid filtering, loading；When loading, chromatographic column liquid outlet is opened, it is to be distilled underwater when moving to column bed surface, it closes
Liquid outlet is added notoginseng polysaccharide solution along column wall with dropper, opens liquid outlet, sample liquid is allowed all to flow into column interior, and
With a small amount of distilled water flushing column wall, then row elution.
Any of the above-described scheme is preferably, and the specific method of the elution is：Use the NaCl of water and various concentration molten respectively
Liquid gradient elution, Anthrone-sulfuricacid method detection make the friendship of DEAE Sepharose Fast Flow anion up to the detection of no polysaccharide
Chromatographic column elution curve is changed, merges each eluting peak solution respectively, neutral notoginseng polysaccharide is eluted with water, and acid notoginseng polysaccharide is not with
It is eluted with concentration NaCl solution.
Any of the above-described scheme is preferably, and the specific method of the dialysis is：Respectively by the eluent after concentration loaded on saturating
It analyses in bag, clamps bag filter both ends with dialysis clamp, be put in distilled water the 36h that dialyses, a water is changed per 4h.
Any of the above-described scheme is preferably, and the bag filter needs to be cut into segment before the use, and every section of about 10cm is put in
30min is boiled in distilled water, boils 10min after washing three times again, later distilled water wash clean；2%NaHCO is used for the second time3With
1mmol/L EDTA are cleaned after boiling.
Any of the above-described scheme is preferably, and the specific method of the freeze-drying is：Eluent is collected in centrifuge tube ,-
40 DEG C of pre-freeze 12h are put in freeze drier freeze-drying 36h-38h, are added after taking-up and about distill water dissolution, shaken up；- 40 DEG C pre-
Freeze 12h, is put in freeze drier for 24 hours；Sealing preserves in drier after taking-up.Freeze drier is cold using FreeZone 4.5
Lyophilizer.Vacuum degree：0.05-0.07MBar；Temperature：-40℃.
Any of the above-described scheme is preferably, and further includes that (3) measure three using High Performance Gel Permeation Chromatography after step (2)
Point of seven Thick many candies, neutrality notoginseng polysaccharide PNPS I and acid notoginseng polysaccharide PNPS II, PNPS III, PNPS IV, PNPS V
Son amount.
Any of the above-described scheme is preferably, and the specific assay method of molecular weight is：
1. standard solution is prepared：Dextran T serial standards：Dextran D0, D1, D2, D3, D4, D5, D6, D7,
D8, D2000 take each standard items respectively, and flowing phased soln is added, and through membrane filtration, filtrate are taken to inject liquid chromatograph, record color
Spectrogram draws standard polysaccharide molecular weight-elution volume standard curve；
2. test solution is prepared：Radix Notoginseng Thick many candies, neutral notoginseng polysaccharide PNPS I, acidity notoginseng polysaccharide II-V are through filter membrane
Sample introduction is filtered, chromatogram is recorded, reference standard curve calculates molecular weight；
3. according to the liquid chromatogram of Series Molecules amount dextran standard items, according to point for the sample that standard curve calculates
Son amount.
Any of the above-described scheme is preferably, and the weight average molecular weight Mw of the neutrality notoginseng polysaccharide PNPS I is 31590Da, number
Average molecular weight Mn is 11170, and polydispersity coefficient D is 2.828.
Any of the above-described scheme is preferably, and further includes that (4) Anthrone-sulfuricacid method measurement Radix Notoginseng Thick many candies are total after step (3)
Sugared content, neutrality notoginseng polysaccharide PNPS I and acid notoginseng polysaccharide II-V contents, DNS methods measure reduced sugar in Radix Notoginseng Thick many candies
Content.
Any of the above-described scheme is preferably, the Radix Notoginseng Thick many candies content=total sugar content-content of reducing sugar.
Any of the above-described scheme is preferably, and the content of the neutrality notoginseng polysaccharide PNPS I is 93.6%.
The present invention also provides a kind of neutral notoginseng polysaccharides prepared by the method for extraction and purification of above-mentioned neutral notoginseng polysaccharide to promote
Application in terms of periodontal ligament stem cell, osteoblast and application on human skin epidermal cell proliferation.
The present invention also provides a kind of neutral notoginseng polysaccharide applications prepared by the method for extraction and purification of above-mentioned neutral notoginseng polysaccharide
In pharmaceutical intermediate.
The present invention also provides a kind of neutral notoginseng polysaccharide applications prepared by the method for extraction and purification of above-mentioned neutral notoginseng polysaccharide
In health food.
The present invention also provides a kind of neutral notoginseng polysaccharide applications prepared by the method for extraction and purification of above-mentioned neutral notoginseng polysaccharide
In daily chemical products.
The present invention also provides toothpaste prepared by neutral notoginseng polysaccharide PNPS I prepared by a kind of above method, by stocking up,
Mixing, grinding, de-airing step and obtain lotion of toothpaste, it is characterised in that：Include the component of following weight percent：Neutral Radix Notoginseng
Polysaccharide PNPS I0.1-2.0%, rubbing agent 2~60%, wetting agent 2~55%, adhesive 0.2~2%, foaming agent 1~
3.5%, surplus is deionized water.
Preferably, further include essence 0.5~2, saccharin sodium 0.2~3.5%, sodium benzoate 0.1~0.3%, edible color
One or more of element 0~2%.
Any of the above-described scheme is preferably, and the rubbing agent is calcium phosphate dibasic dihydrate, silica, calcium carbonate or hydrogen-oxygen
Change one or more of aluminium.
Any of the above-described scheme is preferably, and the wetting agent is one or more of glycerine, sorbic alcohol or polyethylene glycol.
Any of the above-described scheme is preferably, described adhesive be sodium carboxymethylcellulose, hydroxyethyl cellulose, xanthans,
One or more of guar gum or carragheen.
Any of the above-described scheme is preferably, the foaming agent is laruyl alcohol sodium sulfovinate, in sodium lauroyl sarcosine extremely
Few one kind.
The present invention also provides facial masks prepared by neutral notoginseng polysaccharide PNPS I prepared by a kind of above method, using face paper
It immerses in facial mask stoste and obtains, facial mask stoste uses Radix Notoginseng neutral polysaccharide PNPS I, and water is added to be configured to a concentration of 0.5-2mg/mL
Radix Notoginseng neutral polysaccharide PNPS I solution to obtain the final product.
The present invention also provides a kind of craft prepared by neutral notoginseng polysaccharide PNPS I prepared by above-mentioned method for extraction and purification
Soap includes the raw material of following parts by weight：Neutral notoginseng polysaccharide PNPS I2-4g, palm oil 180-200g, coconut oil 70-90g, olive
Olive oil 70-90g, Sweet Almond Oil 70-90g, wheat-germ oil 50-70g, sodium hydroxide 60-80g, distilled water 130-150g, gold and silver
Flower essential oil 0.2-3g.
Preferably, include the raw material of following parts by weight：Neutral notoginseng polysaccharide PNPS I1-2g, palm oil 190g, coconut
Oily 85g, olive oil 80g, Sweet Almond Oil 80g, wheat-germ oil 65g, sodium hydroxide 72g, distilled water 144g, honeysuckle essential oil
0.5-2g。
Any of the above-described scheme is preferably, and includes the raw material of following parts by weight：Neutral notoginseng polysaccharide PNPS I1.5g, palm
Oily 190g, coconut oil 85g, olive oil 80g, Sweet Almond Oil 80g, wheat-germ oil 65g, sodium hydroxide 72g, distilled water 144g,
Honeysuckle essential oil 1g.
The method that the present invention also provides a kind of to prepare handmade soap using neutrality notoginseng polysaccharide PNPS I, preparation method include with
Lower step：
(1) sodium hydroxides are added in distilled water and dissolve, and neutral notoginseng polysaccharide PNPS I are then stirred heat of solution, cooling
To 40 DEG C -50 DEG C, constant temperature is kept, it is spare；
(2) palm oils, coconut oil, olive oil, Sweet Almond Oil, wheat-germ oil mixing are placed in beaker, heating water bath
It is uniformly mixed, the solution that step (1) obtains slowly is poured into, keep 40 DEG C -50 DEG C of bath temperature and quickly stir；
(3) honeysuckle essential oils are added in the mixture that step (2) obtains and are sufficiently stirred, until being uniformly mixed；
(4) pours into the mixture that step (3) obtains in ready mold, until soap blank solidifies completely；
(5) demoulds soap blank in step (4) to obtain notoginseng polysaccharide handmade soap finished product.
Preferably, 45 DEG C are cooled in step (1).
Any of the above-described scheme is preferably, and 45 DEG C of bath temperature is kept in step (2) and is quickly stirred with electric mixer,
Mixture starts to be become cloudy by clarification, continues mixing soap lye being stirred to thick state.
The present invention, using water extraction and alcohol precipitation method, obtains neutrality using the Radix Notoginseng waste residue for extracting notoginsenoside in producing as raw material
Radix Notoginseng Thick many candies, neutral notoginseng polysaccharide content can reach 93.6%.It is handed over using DEAE Sepharose Fast Flow anion
Chromatographic purifying Radix Notoginseng Thick many candies are changed, 5 components are obtained, through dialysis purification, neutral notoginseng polysaccharide PNPS I are obtained after freeze-drying
And 5 acid notoginseng polysaccharide PNPS II, PNPS III, PNPS IV, PNPS V samples.Measure Radix Notoginseng Thick many candies, neutral Radix Notoginseng
Polysaccharide PNPS I and acid notoginseng polysaccharide II-V (acid notoginseng polysaccharide PNPS II, acid notoginseng polysaccharide PNPS III, acidity three
Seven polysaccharide PNPS IV, acidity notoginseng polysaccharide PNPS V) content and molecular weight ranges, study its to Periodontal ligament stem cell and
The influence of mouse bone-forming cell and application on human skin epidermal cell in-vitro multiplication, it was demonstrated that neutral notoginseng polysaccharide PNPS I promote parodontium
Stem cell and mouse bone-forming cell and the effect of application on human skin epidermal cell proliferation are most apparent, and neutral notoginseng polysaccharide can PNPS I conducts
The important drugs intermediate of developing new drug, the natural material of functional health-care food, and also the activity for being alternatively arranged as cosmetics is former
Material.Radix Notoginseng Thick many candies, neutrality notoginseng polysaccharide PNPS I and acidity notoginseng polysaccharide II-V to Periodontal ligament stem cell and mouse at
Osteocyte MC3T3-E1 and application on human skin epidermal cell in-vitro multiplication activity experiment the result shows that：Neutral notoginseng polysaccharide PNPS I exist
Concentration is higher than 35ug/ml, can promote Periodontal ligament stem cell and mouse bone-forming cell MC3T3-E1 and application on human skin epidermis in vitro
Cell is proliferated and is better than Radix Notoginseng Thick many candies and acid notoginseng polysaccharide II-V to the increment of three of the above cell effect, therefore in selecting
Important drugs intermediates of the property notoginseng polysaccharide PNPS I as developing new drug, the natural material of functional health-care food, but also
It can be used as the activated feedstock of cosmetics.
Description of the drawings
Fig. 1 is Radix Notoginseng Thick many candies DEAE Sepharose Fast Flow anion-exchange chromatography 0-2mol/LNaCl solution
Gradient elution curve；
Fig. 2 is Radix Notoginseng Thick many candies HPGPC chromatograms；
Fig. 3 is neutral notoginseng polysaccharide PNPSI HPGPC chromatograms；
Fig. 4 is acid notoginseng polysaccharide PNPSII HPGPC chromatograms；
Fig. 5 is acid notoginseng polysaccharide PNPSIII HPGPC chromatograms；
Fig. 6 is acid notoginseng polysaccharide PNPSIV HPGPC chromatograms；
Fig. 7 is acid notoginseng polysaccharide PNPSV HPGPC chromatograms.
Specific implementation mode
In order to further appreciate that the technical characteristic of the present invention, the present invention is carried out with reference to specific drawings and examples detailed
Carefully illustrate.Embodiment only has illustrative effect to the present invention, without the effect of any restrictions, the skill of this field
The modification for any unsubstantiality that art personnel make on the basis of the present invention, should all belong to the scope of protection of the present invention.
Embodiment 1：
A kind of method for extraction and purification of neutrality notoginseng polysaccharide, specifically includes following steps：
(1) extraction of Radix Notoginseng Thick many candies：The waste residue 1kg for extracting arasaponin is weighed, 10 times of amount water are added for the first time,
Boiling water bath boils 8h, collects filtrate, second of addition, 5 times of amount water, boiling water bath 8h.Merging filtrate twice, 4500rpm centrifuges 5min,
Take supernatant.80 DEG C of spin concentrations are stood for 24 hours to the 1/8 of original volume, and centrifugation abandons precipitation, is slowly added to three times absolute ethyl alcohol, side
Edged stirs, and 4 DEG C of placement 12h filter to obtain precipitation.75% ethyl alcohol washing precipitation for several times, is used appropriate water-soluble after ethyl alcohol volatilization is done
Supernatant is collected in solution, centrifugation, and three times absolute ethyl alcohol is added, and 4 DEG C are placed 12h, filters to obtain precipitation, 75% ethyl alcohol washing precipitation, 50 DEG C
Baking oven is dried to get Radix Notoginseng Thick many candies.
(2) DEAE Sepharose Fast Flow anion-exchange chromatographies purify Thick many candies
1. filling column：.Wet method dress post, pillar fill in cotton, and distilled water is added in column and a bit of liquid level, glass bar is kept to press
Pressure cotton dispels bubble.It is poured slowly into column along column wall with glass bar guiding homogenate, bubble cannot be generated.It opens pillar and goes out liquid
Mouthful, make gel free settling in column, washes with water chromatographic column edge.There cannot be tomography when filling column, then use distillation water balance,
Column specification is 2.4 × 40cm.
2. balancing：Distilled water crosses 0.45 μm of filter membrane, pillar is rinsed with 5 times of cylinder ponding with certain flow rate, until efflux
Conductivity and pH are constant, balance 48 hours.
3. loading：Radix Notoginseng Thick many candies are dissolved in a small amount of distilled water, are configured to 30mg/mL, and 4500rpm centrifuges 10min, supernatant
0.45 μm of filter filtering of liquid, loading.When loading, chromatographic column liquid outlet is opened, it is to be distilled underwater when moving to column bed surface, it closes
Liquid outlet is added notoginseng polysaccharide solution along column wall with dropper, opens liquid outlet, sample liquid is allowed all to flow into column interior, and
With a small amount of distilled water flushing column wall, then row elution.
4. eluting：Respectively with 0,0.1,0.2,0.3,0.4,0.5,2mol/L NaCl solution gradient elutions, each elution about 5
Times column volume, flow velocity 0.8mL/min, often pipe meet 2mL, Anthrone-sulfuricacid method detection, until the detection of no polysaccharide, to elute pipe number
For abscissa, absorbance value is that ordinate makees DEAE Sepharose Fast Flow anion exchange chromatography elution curves
Figure, merges each eluting peak solution, is concentrated under reduced pressure into 1/10 volume respectively.Part is eluted with water as neutral notoginseng polysaccharide；Remaining
It is acid notoginseng polysaccharide with various concentration NaCl solution elution fraction.Radix Notoginseng Thick many candies DEAE Sepharose Fast Flow
Anion-exchange chromatography 0-2mol/LNaCl solution gradient elution curves are shown in Figure of description 1.
5. dialysing：Bag filter is cut into every section of about 10cm, is put in distilled water and boils 30min, boiled again after washing three times
10min, distilled water wash clean.Bag filter can use three times, use 2%NaHCO for the second time3It is clear after being boiled with 1mmol/L EDTA
It washes.Respectively by bag filter of the eluent after concentration loaded on molecular weight 14000, about 1/2 volume clamps bag filter with dialysis clamp
Both ends are put in distilled water the 36h that dialyses, and a water is changed per 4h.
6. being freeze-dried：Polysaccharide the meeting moisture absorption, notoginseng polysaccharide eluent lyophilized technique in freezing dry process：By eluent
50mL centrifuge tubes are collected in, about 16mL is filled, -40 DEG C of pre-freeze 12h are put in freeze drier 36h, and about 1mL distillations are added after taking-up
Water dissolution shakes up.- 40 DEG C of pre-freeze 12h, are put in freeze drier for 24 hours.Sealing preserves in drier after taking-up.
(3) molecular weight determination
1. standard solution is prepared：Dextran T serial standards：Dextran D0, D1, D2, D3, D4, D5, D6, D7,
D8, D2000, standard molecular weight is respectively 180,2500,4600,7100,10000,21400,41100,84400,133800,
2000000Da.Each standard items 10mg is taken respectively, and 1ml mobile phases (0.1mol/L NaCl) is added to dissolve, through 0.22 μm of membrane filtration,
20 μ L injection liquid chromatographs of filtrate are taken, chromatogram is recorded, draws standard polysaccharide molecular weight-elution volume standard curve.
2. test solution is prepared：Radix Notoginseng Thick many candies, neutral notoginseng polysaccharide PNPS I, acidity notoginseng polysaccharide II-V (10mg/
ML) through 0.22 μm of membrane filtration sample introduction, chromatogram is recorded, reference standard curve calculates molecular weight.
3. according to the liquid chromatogram of Series Molecules amount dextran standard items, the equation drawn is:
log Mw=-0.7920V+12.1438, R2=0.9761.
Molecular weight according to the sample of standard curve calculating is as shown in table 1, Radix Notoginseng Thick many candies, neutrality notoginseng polysaccharide PNPS I
And acid notoginseng polysaccharide PNPS II, PNPS III, PNPS IV, PNPS V chromatograms are shown in Figure of description 2- attached drawings 7.
1 Radix Notoginseng Thick many candies of table, neutrality notoginseng polysaccharide PNPS I and acid notoginseng polysaccharide PNPS II~V molecular weight determination knots
Fruit
From the above results, neutral notoginseng polysaccharide PNPS I weight average molecular weight is 31590, molecular weight distribution spread factor
D is 2.828, respectively less than other groups, shows neutral polysaccharide than other components sub- amount narrowly distributing.
(4) Anthrone-sulfuricacid method measures notoginseng polysaccharide content
Anthrone-sulfuricacid method measure Radix Notoginseng Thick many candies total sugar content, DNS methods measure Radix Notoginseng Thick many candies in content of reducing sugar, three
The polyoses content of seven Thick many candies contents=total sugar content-content of reducing sugar, Radix Notoginseng Thick many candies is 63.86%；Anthrone-sulfuricacid method is surveyed
Fixed neutrality notoginseng polysaccharide PNPS I and acid notoginseng polysaccharide II-V contents are shown in Table 2.
2 neutrality notoginseng polysaccharide PNPS I of table and acid notoginseng polysaccharide PNPS II~V polyoses contents and yield
As can be seen from the above table, neutral notoginseng polysaccharide PNPS I content highests, up to 93.6%.
2 notoginseng polysaccharide of embodiment is thin to Periodontal ligament stem cell and mouse bone-forming cell MC3T3-E1 and application on human skin epidermis
The outer proliferative effect experiment of cell space
One, mtt assay measures influence of the notoginseng polysaccharide to Periodontal ligament stem cell in-vitro multiplication
Using regular growth cultivation culture cell.The Periodontal ligament stem cell for taking 7 generation exponential phases is adjusted thin after counting
Born of the same parents' concentration is inoculated in 96 orifice plates, and 100/hole, every 90 μ l, CO2 incubators culture of hole adds PNPS I, PNPS respectively after 12 hours
II, PNPS III, PNPS IV and each 10 μ l of Radix Notoginseng Thick many candies solution, make to act on the final concentration of 6.72mg/ml of cell,
4.48mg/ml, 2.24mg/ml, 1.12mg/ml, 560ug/ml, 280ug/ml, 140ug/ml, 70ug/ml, each concentration set 6
A parallel hole, negative control are isometric MEM-ALPHA culture solutions, add 10 μ l of physiological saline.After 48 hours, add MTT solution
(5mg/ml) 20 μ l continue culture 4 hours per hole, and 150 μ lDMSO are added per hole and fully dissolve, each hole is measured with microplate reader
A570 values.The OD values of the A570 of various concentration are calculated, experimental result is shown in Table 3：
Influence experimental result (n=6) of 3 notoginseng polysaccharide of table to Periodontal ligament stem cell in-vitro multiplication
Note：*P<0.05；**P<0.01
Neutral notoginseng polysaccharide PNPS I has promotion to make in 35~1120ug/ml of concentration, to the proliferation of Periodontal ligament stem cell
With；PNPS II (acidic polysaccharose) have facilitation in 17.5~70ug/ml of concentration, to the proliferation of Periodontal ligament stem cell；PNPS
III (acidic polysaccharose) and PNPS IV (acidic polysaccharose) have inhibiting effect to the proliferation of Periodontal ligament stem cell；Radix Notoginseng Thick many candies exist
Concentration 70ug/ml has facilitation to the proliferation of Periodontal ligament stem cell.Neutral notoginseng polysaccharide PNPS I is dry to people's parodontium thin
The Effect of promoting growth of born of the same parents is most apparent.
Two, mtt assay measures influence of the notoginseng polysaccharide to mouse bone-forming cell MC3T3-E1MTT in-vitro multiplications
Using regular growth cultivation culture cell.The mouse bone-forming cell for taking 7 generation exponential phases, cell is adjusted after counting
Concentration is inoculated in 96 orifice plates, and 100/hole, every 90 μ l, CO2 incubators culture of hole adds PNPS I, PNPS respectively after 12 hours
II, PNPS III, PNPS IV and each 10 μ l of Radix Notoginseng Thick many candies solution, make to act on the final concentration of 1.12mg/ml of cell,
560ug/ml, 280ug/ml, 140ug/ml, 70ug/ml, 35ug/ml, 17.5ug/ml, each concentration set 6 parallel holes, negative
Control is isometric MEM-ALPHA culture solutions, adds 10 μ l of physiological saline.After 48 hours, add 20 μ l of MTT solution (5mg/ml) every
Culture 4 hours is continued in hole, and 150 μ lDMSO are added per hole and fully dissolve, the A570 values in each hole are measured with microplate reader.It calculates different
The OD values of the A570 of concentration, experimental result are shown in Table 4：
4 notoginseng polysaccharide of table influences experimental result (n=6) to mouse bone-forming cell MC3T3-E1 in-vitro multiplications
Note：*P<0.05；**P<0.01
Neutral notoginseng polysaccharide PNPS I, PNPS II (acidic polysaccharose), PNPS III (acidic polysaccharose), PNPS IV are (acid
Polysaccharide) and Radix Notoginseng Thick many candies have a facilitation to mouse bone-forming cell MC3T3-E1 in-vitro multiplications, in the results show
Property notoginseng polysaccharide PNPS I facilitations are most apparent.
Three, influence of the notoginseng polysaccharide to application on human skin epidermal cell in-vitro multiplication is measured
Culture people's normal skin epidermal cell (is radically reformed vast and boundless biotechnology company) purchased from Beijing, culture medium Eplife
Add the HKGS assisting growth factors, the next day change liquid.Cell passes on after covering with bottle, and adjustment cell concentration is 1 × 105/ml,
96 hole flat undersides are inoculated in, per 100 μ l of hole, are placed in 37 DEG C, 5%CO2It is cultivated in incubator.Cell is divided into 10 groups, every group is 6
Hole.After cell culture for 24 hours, every group of cell is separately added into plus PNPS I, PNPS II, PNPS III, PNPS IV and Radix Notoginseng are thick
Each 10 μ l of polysaccharide solution, make to act on final concentration of 1.12mg/ml, 560ug/ml of cell, 280ug/ml, 140ug/ml,
70ug/ml, 35ug/ml, 17.5ug/ml, 100 μ l of cultured epidermal cell liquid, negative control group be directly added into epidermal cell training
100 μ l of nutrient solution.After continuing culture for 24 hours, CCK8 solution is added, per 20 μ l of hole, continues to cultivate 2.5h, it is measured with enzyme mark detector
Absorbance of cells (OD value wavelength 450nm, reference wavelength 600nm), experimental result is shown in Table 5：
Influence experimental result (n=6) of 5 notoginseng polysaccharide of table to application on human skin epidermal cell in-vitro multiplication
Note：*P<0.05；**P<0.01
Neutral notoginseng polysaccharide PNPS I have promotion in 35~1120ug/ml of concentration, to application on human skin epidermal cell in-vitro multiplication
Effect；PNPS II (acidic polysaccharose) have facilitation in 35~1120ug/ml of concentration, to application on human skin epidermal cell in-vitro multiplication；
Radix Notoginseng Thick many candies (acidic polysaccharose) have facilitation in 140~1120ug/ml of concentration, to application on human skin epidermal cell in-vitro multiplication；
PNPS III (acidic polysaccharose) and PNPS IV (acidic polysaccharose) are on application on human skin epidermal cell in-vitro multiplication without influence.
Above-mentioned experiment, research Radix Notoginseng Thick many candies, neutrality notoginseng polysaccharide PNPS I and acid notoginseng polysaccharide II-V (acid Radix Notoginseng
Polysaccharide PNPS II, PNPS III, PNPS IV, PNPS V) to Periodontal ligament stem cell and mouse bone-forming cell and application on human skin
The influence of epidermal cell in-vitro multiplication, the results show neutrality notoginseng polysaccharide PNPS I promote periodontal ligament stem cell and mouse at
Osteocyte and the effect of application on human skin epidermal cell proliferation are most apparent.
Radix Notoginseng Thick many candies, neutrality notoginseng polysaccharide PNPS I and acidity notoginseng polysaccharide II-V are to Periodontal ligament stem cell and small
Mouse osteoblast MC3T3-E1 and application on human skin epidermal cell in-vitro multiplication activity experiment are the result shows that neutral notoginseng polysaccharide PNPS
I can promote the proliferation of Periodontal ligament stem cell in a concentration of 35ug/ml~1120ug/ml；A concentration of 35ug/ml~
1120ug/ml can promote the proliferation of mouse bone-forming cell MC3T3-E1；In a concentration of 35ug/ml~1120ug/ml, can promote
Application on human skin epidermal cell proliferation is better than Radix Notoginseng Thick many candies and acid notoginseng polysaccharide II-V to the increment effect of three of the above cell,
Therefore select important drugs intermediates of the neutrality notoginseng polysaccharide PNPS I as developing new drug, the natural original of functional health-care food
Material, and it is alternatively arranged as the activated feedstock of cosmetics.
Embodiment 3
It is raw material with content highest and the promotion most significant Radix Notoginseng neutral polysaccharide PNPS I of cell-proliferation activity, prepares Radix Notoginseng
Polysaccharide toothpaste.
Present embodiment discloses a kind of toothpaste and its preparation method and application of the PNPS of neutral polysaccharide containing Radix Notoginseng I.Radix Notoginseng is more
Sugared toothpaste includes Radix Notoginseng neutral polysaccharide PNPS I and rubbing agent, wetting agent, adhesive, foaming agent, essence, saccharin sodium, benzoic acid
Sodium, food coloring, deionized water.Active constituent in toothpaste is weight percentage as the Radix Notoginseng neutral polysaccharide of 0.1-2.0%
PNPS I；Rubbing agent is to account for the calcium phosphate dibasic dihydrate, silica, calcium carbonate, hydrogen-oxygen that toothpaste weight percentage is 2~60%
Change one or more of aluminium；Wetting agent is to account in the glycerine, sorbic alcohol, polyethylene glycol that toothpaste weight percentage is 2~55%
One or more；Adhesive be account for sodium carboxymethylcellulose, hydroxyethyl cellulose that toothpaste weight percentage is 0.2~2%,
The one or more of xanthans, guar gum, carragheen；Foaming agent is to account for the laruyl alcohol sulphur that toothpaste weight percentage is 1~3.5%
The one or more of acid esters sodium (lauryl sodium sulfate), sodium lauroyl sarcosine (dodecanoyl sodium sarcosinate)；Essence accounts for tooth
Cream weight percent is 0.5~2%；It is 0.2~3.5% that saccharin sodium, which accounts for toothpaste weight percentage,；Sodium benzoate accounts for toothpaste weight
Percentage is 0.1~0.3%；It is 0~2% that toothpaste accounts for toothpaste weight percentage with pigment；Surplus is deionized water.
The preparation method of notoginseng polysaccharide toothpaste include stock, mixing, grinding, de-airing step and obtain lotion of toothpaste.
In addition to cleaning the teeth, the present invention has prevention periodontitis, gingivitis, bleeding gums, canker sore, prolongs notoginseng polysaccharide toothpaste
The effect of slow gingival atrophy.It, will not be to oral cavity just since the Radix Notoginseng neutral polysaccharide PNPS I of the present invention are natural plant extracts
Normal flora ecological environment causes brokenly meeting, and the formation that can promote oral cavity ideal micropopulation is used for a long time, and enhances human body exogenous
The resistivity of pathogen improves oral healthy condition.
Embodiment 4
It is raw material with content highest and the promotion most significant Radix Notoginseng neutral polysaccharide PNPS I of cell-proliferation activity, prepares Radix Notoginseng
Polysaccharide facial mask.
Present embodiment discloses a kind of facial masks and its preparation method and application of the PNPS of neutral polysaccharide containing Radix Notoginseng I.Facial mask is adopted
It is soaked in neutral notoginseng polysaccharide facial mask stoste and is made with face paper.Neutral notoginseng polysaccharide facial mask stoste described in the embodiment
It is appropriate to be prepared as weighing Radix Notoginseng neutral polysaccharide PNPS I, water is added to be configured to a concentration of 0.5-2mg/mL Radix Notoginseng neutral polysaccharide PNPS I
Solution to obtain the final product.The present invention can promote reparation, proliferation and the activity of face cell, keep skin more flexible.
Embodiment 5
It is raw material with content highest and the promotion most significant Radix Notoginseng neutral polysaccharide PNPS I of cell-proliferation activity, prepares Radix Notoginseng
Polysaccharide handmade soap.
Neutrality notoginseng polysaccharide handmade soap provided in this embodiment, including following raw material：Neutral notoginseng polysaccharide PNPS I1-2g,
Palm oil 190g, coconut oil 85g, olive oil 80g, Sweet Almond Oil 80g, wheat-germ oil 65g, sodium hydroxide 72g, distilled water
144g, honeysuckle essential oil 0.5-2g.
Its preparation process is：
(1) required sodium hydroxide is slowly added into distilled water and is dissolved, is stirred to always until solution becomes transparent, then
Notoginseng polysaccharide is stirred into heat of solution, placement makes it be cooled to 45 DEG C, keeps the temperature, spare；
(2) palm oil, coconut oil, olive oil, Sweet Almond Oil, wheat-germ oil are mixed and is placed in beaker, 45 DEG C of water
Bath heating is uniformly mixed, and the solution in step (1) is slowly poured into, saponification occurs in grease, keeps 45 DEG C of bath temperature simultaneously
It is quickly stirred with electric mixer, mixture starts to be become cloudy by clarification, continues mixing soap lye being stirred to thick state；
(3) honeysuckle essential oil is added in step (2) mixture and is sufficiently stirred, until being uniformly mixed；
(4) stop stirring, the mixture that step (3) obtains is poured into ready mold after slightly cooling, is placed in the moon
Cool ventilation drying, until soap blank solidifies completely；
(5) soap blank demoulding in step (4) is just obtained into notoginseng polysaccharide handmade soap finished product.
Claims (17)
1. a kind of neutrality notoginseng polysaccharide is preparing promotion periodontal ligament stem cell, osteoblast and application on human skin epidermal cell proliferation side
The application of the drug in face, the neutrality notoginseng polysaccharide use the Radix Notoginseng waste residue for extracting notoginsenoside for raw material, it is characterised in that：
Include the following steps：
(1) water extraction and alcohol precipitation method extracts Radix Notoginseng Thick many candies；
(2) DEAE Sepharose Fast Flow anion-exchange chromatographies and dialysis purify Thick many candies, and freeze-drying obtains
To neutral notoginseng polysaccharide PNPS I and acid notoginseng polysaccharide PNPS II, acid notoginseng polysaccharide PNPS III, acid notoginseng polysaccharide
PNPS IV, acid notoginseng polysaccharide PNPS V freeze-dried powders.
2. the application of neutrality notoginseng polysaccharide according to claim 1, it is characterised in that：Water extract-alcohol precipitation in the step (1)
Method extracts Radix Notoginseng Thick many candies specific method：The waste residue for extracting arasaponin is taken, water is added, boiling water bath boils, and collects filtrate,
Centrifugation, takes supernatant；After spin concentration, standing, centrifugation abandons precipitation, is slowly added to absolute ethyl alcohol, stirring while adding, after placement,
Filter to obtain precipitation；Ethyl alcohol washing precipitation, is collected supernatant, anhydrous second is added after ethyl alcohol volatilization is done with appropriate water dissolution, centrifugation
Alcohol after placement, filters to obtain precipitation, ethyl alcohol washing precipitation, and baking oven is dried to get Radix Notoginseng Thick many candies.
3. the application of neutrality notoginseng polysaccharide according to claim 1, it is characterised in that：DEAE in the step (2)
Sepharose Fast Flow anion-exchange chromatographies and dialysis purifying Thick many candies method include dress column, balance, loading,
Elution, dialysis and freeze-drying.
4. the application of neutrality notoginseng polysaccharide according to claim 3, it is characterised in that：It is described dress column specific method be：
Ethyl alcohol is added, filters, homogenate is made into water, using wet method dress post, is poured slowly into column along column wall with glass bar guiding homogenate；
Pillar liquid outlet is opened, makes gel free settling in column, washes with water chromatographic column edge.
5. the application of neutrality notoginseng polysaccharide according to claim 4, it is characterised in that：The specific method of the wet method dress post
For：Pillar fills in cotton, and distilled water is added in column and keeps a bit of liquid level, and glass bar pressing cotton dispels bubble.
6. the application of neutrality notoginseng polysaccharide according to claim 3, it is characterised in that：The specific method of the balance is：
Distilled water filter membrane until efflux conductivity and pH are constant, balances 48 hours with more times of amount volume of water to rinse pillar.
7. the application of neutrality notoginseng polysaccharide according to claim 3, it is characterised in that：The specific method of the loading is：
Radix Notoginseng Thick many candies are dissolved in a small amount of distilled water, centrifugation, supernatant liquid filtering, loading；When loading, chromatographic column liquid outlet is opened, is waited for
When distilled water is displaced downwardly to column bed surface, liquid outlet is closed, notoginseng polysaccharide solution is added along column wall with dropper, opens liquid outlet,
It allows sample liquid all to flow into column interior, a small amount of distilled water flushing column wall is used in combination, is then eluted.
8. the application of neutrality notoginseng polysaccharide according to claim 3, it is characterised in that：The specific method of the elution is：
The NaCl solution gradient elution of water and various concentration, Anthrone-sulfuricacid method detection is used to make DEAE up to the detection of no polysaccharide respectively
Sepharose Fast Flow anion exchange chromatography elution curves, merge each eluting peak solution, neutral Radix Notoginseng is more respectively
Sugar is eluted with water, and acid notoginseng polysaccharide is eluted with various concentration NaCl solution.
9. the application of neutrality notoginseng polysaccharide according to claim 3, it is characterised in that：The specific method of the dialysis is：
The eluent after concentration is clamped bag filter both ends with dialysis clamp, is put in distilled water the 36h that dialyses loaded in bag filter respectively,
A water is changed per 4h.
10. the application of neutrality notoginseng polysaccharide according to claim 9, it is characterised in that：The bag filter is before the use
It needs to be cut into segment, every section of 10cm is put in distilled water and boils 30min, boils 10min after washing three times again, later distillation washing
Totally；2%NaHCO is used for the second time3It is cleaned after being boiled with 1mmol/L EDTA.
11. the application of neutrality notoginseng polysaccharide according to claim 3, it is characterised in that：The specific side of the freeze-drying
Method is：Eluent is collected in centrifuge tube, -40 DEG C of pre-freeze 12h are put in freeze drier freeze-drying 36h-38h, add after taking-up
Enter to distill water dissolution, shake up；- 40 DEG C of pre-freeze 12h, are put in freeze drier for 24 hours；Sealing preserves in drier after taking-up.
12. the application of neutrality notoginseng polysaccharide according to claim 1, it is characterised in that：Further include step after step (2)
(3) High Performance Gel Permeation Chromatography is used to measure Radix Notoginseng Thick many candies, neutrality notoginseng polysaccharide PNPS I and acid notoginseng polysaccharide PNPS
The molecular weight of II, PNPS III, PNPS IV.
13. the application of neutrality notoginseng polysaccharide according to claim 12, it is characterised in that：The specific assay method of molecular weight
For：
1. standard solution is prepared：Dextran T serial standards：Dextran D0, D1, D2, D3, D4, D5, D6, D7, D8,
D2000 takes each standard items respectively, and flowing phased soln is added, and through membrane filtration, filtrate is taken to inject liquid chromatograph, records chromatography
Figure draws standard polysaccharide molecular weight-elution volume standard curve；
2. test solution is prepared：Radix Notoginseng Thick many candies, neutral notoginseng polysaccharide PNPS I, acidity notoginseng polysaccharide II-V are through membrane filtration
Sample introduction, records chromatogram, and reference standard curve calculates molecular weight；
3. according to the liquid chromatogram of Series Molecules amount dextran standard items, the molecular weight of sample is calculated according to standard curve.
14. the application of neutrality notoginseng polysaccharide according to claim 12, it is characterised in that：The neutrality notoginseng polysaccharide PNPS
The weight average molecular weight Mw of I is 31590, and number-average molecular weight Mn is 11170, and polydispersity coefficient D is 2.828.
15. the application of neutrality notoginseng polysaccharide according to claim 12, it is characterised in that：Further include (4) after step (3)
Anthrone-sulfuricacid method measurement Radix Notoginseng Thick many candies total sugar content, neutrality notoginseng polysaccharide PNPS I and acid notoginseng polysaccharide PNPS II,
Acid notoginseng polysaccharide PNPS III, acid notoginseng polysaccharide PNPS IV, acid notoginseng polysaccharide PNPS V contents, DNS methods measure Radix Notoginseng
Content of reducing sugar in Thick many candies.
16. the application of neutrality notoginseng polysaccharide according to claim 15, it is characterised in that：The Radix Notoginseng Thick many candies content=
Total sugar content-content of reducing sugar.
17. the application of neutrality notoginseng polysaccharide according to claim 15, it is characterised in that：The neutrality notoginseng polysaccharide PNPS
The content of I is 93.6%.
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| CN107653227A (en) * | 2017-11-13 | 2018-02-02 | 重庆斯德姆生物技术有限公司 | A kind of inducing culture and abductive approach for promoting Differentiation of Marrow Stromal Stem Cells Into Neurons |
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