CN104262504B - The preparation of Fructus Phyllanthi polysaccharide and application - Google Patents
The preparation of Fructus Phyllanthi polysaccharide and application Download PDFInfo
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- CN104262504B CN104262504B CN201410574230.9A CN201410574230A CN104262504B CN 104262504 B CN104262504 B CN 104262504B CN 201410574230 A CN201410574230 A CN 201410574230A CN 104262504 B CN104262504 B CN 104262504B
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Abstract
The invention discloses the preparation method of a kind of Fructus Phyllanthi polysaccharide, its preparation process includes that extracting of Fructus Phyllanthi polysaccharide separates and the decoloration process research of Fructus Phyllanthi crude polysaccharides, it is characterized in that Fructus Phyllanthi crude polysaccharides uses insoluble chitosan to be that bleaching agent bleaching refines, it solve in tradition decolorizing and refining or percent of decolourization is low or polyoses content is low shortcoming, the Fructus Phyllanthi polysaccharide using the method to prepare, is all better than 5 fluorouracil (5 FU) to the suppression ratio of hepatoma H22 cells cell and the suppression ratio of esophageal cancer cell strain EC109.
Description
Technical field
The present invention relates to preparation and the application of Fructus Phyllanthi polysaccharide, particularly relate to a kind of tide with anti-alimentary tract tumor activity
The preparation of Shan Fructus Phyllanthi polysaccharide and application.
Background technology
Fructus Phyllanthi Phyllanthus emblica L. has another name called Acronychi apedunculata (L.) Miq., Phyllanthus embical fruit, Phyllanthus emblica L. etc., for Euphorbiaceae Cacumen Securinegae Suffruticosae
The mature fruit of platymiscium Fructus Phyllanthi.Fructus Phyllanthi is one of fine germplasm resources of Chao-Shan Area, and Fructus Phyllanthi not only has abundant
Nutritive value, and there is the multiple biological activitys such as antiinflammatory antioxidation, immunomodulating, antitumor.Current study show that, emblic
The main active of son is: polysaccharose substance, flavone compound, ascorbic acid, SOD enzyme and polyphenol compound, and it is raw
Thing function is the synergistic result of plurality of active ingredients.Wherein, Fructus Phyllanthi polysaccharide (crude polysaccharides) has removing free radical, resists
The effect such as oxidation and antitumor, causes the interest of scholars.
In extracting and developing, purification and the structural research of polysaccharide, decolouring is an important step.At present, decolouring is main
Peroxide passivation to be used and active carbon adsorption, minimum with the percent of decolourization of activated carbon decolorizing, the content of polysaccharide is the highest；Hydrogen peroxide
Good compared with activated carbon of percent of decolourization, but polyoses content is relatively low.Lack a kind of discoloration method quick, simple, efficient at present, both protected
While demonstrate,proving high percent of decolourization, it is ensured that have much higher sugar retention rate.
Simultaneously to the research that the anti-alimentary tract tumor of Fructus Phyllanthi polysaccharide is active.
Summary of the invention
It is an object of the invention to provide the preparation method of a kind of Fructus Phyllanthi polysaccharide, the decolouring essence of particularly thick Fructus Phyllanthi polysaccharide
Method processed, it solves in tradition decolorizing and refining or percent of decolourization is low or polyoses content is low shortcoming.
The present invention is realized by techniques below step, and it is with Fructus Phyllanthi as raw material, follows the steps below system
Standby:
(1) Fructus Phyllanthi enucleation, with 50～80 DEG C of hot-water soaks 10～60 min, then pulls an oar, serosity is heated to reflux 1
～after 5h, hot extract filtered while hot；
(2) gained filtrate being concentrated into original volume 20～30%, being adjusted to solution ethanol content with 95% edible ethanol is 70
～85%, standing 8～15hr, filter, precipitation washes 1～3 time with appropriate 80% ethanol, dehydrated alcohol successively；
(3) precipitation obtains Fructus Phyllanthi crude polysaccharides through filtration, lyophilization.
(4) with water as solvent, Fructus Phyllanthi crude polysaccharides is made into 0.2～0.8 mg/ml solution, with Fructus Citri Limoniae acid for adjusting pH extremely
3.0～4.0, add 2.0～2.5 g insoluble chitosans by every 100 ml solution, make temperature 40 in water-bath～
50 DEG C, stirring 45～60 min decolouring, it is filtered to remove residue.
(5) gained filtrate is concentrated into original volume 20～30%, stands 12-24hr, filter, precipitate freeze-dried obtaining
Fructus Phyllanthi polysaccharide.
In the technology of the present invention step (1), Fructus Phyllanthi is 1:5～1:10 with the ratio of hot water.
In the technology of the present invention step (2), filtrate concentrates and normal pressure can be used to concentrate, it is possible to use concentrating under reduced pressure.
The technology of the present invention step (2) filters and can use centrifugal filtration.
In the technology of the present invention step (2) or step (5), cryogenic temperature is-4 DEG C～-20 DEG C.
PH is preferably adjusted to 3.0～4.0 by the technology of the present invention step (4).
In the technology of the present invention step (4), stirring bleaching temperature is 40 DEG C～50 DEG C.
In the technology of the present invention step (4), stirring bleaching time is 45 min～60min.
The assay method of percent of decolourization of the present invention and polysaccharide retention rate is as follows:
1. percent of decolourization assay method
Prepare 0.5 mg/ml Fructus Phyllanthi polysaccharide sample solution, Fructus Phyllanthi crude polysaccharides solution has been carried out visible ray 400～
600 nm scannings, determine its absorption spectrum.Fructus Phyllanthi crude polysaccharides solution, without maximum absorption band, is decoloured by Fructus Phyllanthi polysaccharide solution
Front is orange-yellow, and according to complementary color principle, solution mainly absorbs blue wave band (446～464nm) visible ray, therefore selects
450 nm are detection wavelength.
Before and after taking appropriate decolouring, liquid to be measured measures absorbance at 450 nm, and (1) calculates percent of decolourization as the following formula:
Percent of decolourization (%)=(ABefore decolouring-AAfter decolouring)/ ABefore decolouring× 100%。
Note: A is absorbance.
2. polysaccharide retention rate assay method
Preparing 0.5 mg/ml Fructus Phyllanthi polysaccharide sample solution, before and after taking decolouring, each 2.00 ml of liquid to be measured, add quality and divide
Number is heavily steaming phenol 1.0 ml and concentrated sulphuric acid 5.0 ml of 6%, after shaking up rapidly, stands 10 min, and boiling water water-bath adds
Hot 15min, is cooled to room temperature, surveys trap A value, calculate the Fructus Vitis viniferae in polysaccharide solution with standard curve at 490 nm wavelength
Sugar content and polysaccharide retention rate.
Polysaccharide retention rate (%)=C × D/W
In formula: W is Fructus Phyllanthi polysaccharide quality (mg);C be glucose quality concentration in polysaccharide solution (mg/
ml) ;D is the extension rate of polysaccharide.
The present invention has good inhibiting effect to anti-tumor activity.
Embodiment
The digestion of HepG2, EC109 cell strain is made the cell suspension of 5 × 10-4/L, the amount of every hole 100 μ L
It is inoculated in 96 well culture plates, puts 37 DEG C, 5% CO2 cell culture incubator with the DMEM culture fluid containing 10% hyclone
Middle cultivation 24h, after requiring to cultivate according to experiment packet, every hole adds 20 μ L MTT (5mg/mL) solution, at 37 DEG C
Continuing to hatch sucking-off supernatant after 4 h, every hole adds 150 μ L DMSO, shakes up with plate shaker, by microplate reader 490
The absorbance (A value) in every hole is detected at nm wavelength.With 5-fluorouracil (5-FU) for contrast, thin to hepatoma H22 cells
The suppression ratio of born of the same parents and the suppression ratio result of esophageal cancer cell strain EC109 are shown in Tables 1 and 2 respectively.It is thin to hepatoma H22 cells
The suppression ratio of born of the same parents and esophageal cancer cell strain EC109 is all better than 5-FU.
The table 1 suppression ratio to hepatoma H22 cells cell
Result: Fructus Phyllanthi polysaccharide is better than 5-FU to the inhibitory activity of hepatoma H22 cells.
The table 2 suppression ratio to esophageal cancer cell strain EC109
Result: Fructus Phyllanthi polysaccharide is better than 5-FU to the inhibitory activity of esophageal cancer cell strain EC109.
Detailed description of the invention
The method provided the present invention below by example is used, but limits the present invention the most in any form.
Embodiment 1
Fructus Phyllanthi enucleation, 60 DEG C of hot-water soak 20 min.Then pull an oar, after being heated to reflux 3h with 1:8 solid-liquid ratio,
Hot extract filtered while hot, centrifugal (3000 r/min, 10 min), it is concentrated in vacuo to original volume 1/4, uses 95% edible wine
Being fine-tuning to solution ethanol content is 80%, stands overnight, centrifugal (3000 r/min, 10 min), and precipitation is used successively
Appropriate 80% ethanol, dehydrated alcohol wash 2 times, and it is standby that vacuum lyophilization obtains Fructus Phyllanthi crude polysaccharides.
Fructus Phyllanthi crude polysaccharides, is configured to 0.5 mg/ml Fructus Phyllanthi polysaccharide sample solution after decolouring.
Embodiment 2
Crude polysaccharides in embodiment is configured to the Fructus Phyllanthi crude polysaccharides solution of 0.5 mg/ml, takes Fructus Phyllanthi polysaccharide sample
Solution 50.00 ml, investigates different discoloration methods.Method one: sample solution adds activated carbon 2%, with Fructus Citri Limoniae acid for adjusting pH extremely
4.0,60 DEG C of stirring in water bath are decoloured 30 min, are filtered to remove residue；Method two: sample solution adds H2O2 solution 12.5ml
(H2O2 solution and polysaccharide solution are than 1:4, V/V), regulates pH to 9. 0 with 1%NaOH or strong aqua ammonia, adds in 60 DEG C of water-baths
Thermal agitation 3 h, then adds heat abstraction hydrogen peroxide；Method three: sample solution adds insoluble chitosan by 1.5 g/100ml,
With Fructus Citri Limoniae acid for adjusting pH to 4.0,60 DEG C of water-bath intermittent stirrings decolour 30 min, are filtered to remove residue.Measure respectively and calculate
Percent of decolourization and polyoses content, the results are shown in Table 3.
The different discoloration method of table 3 compares
From table 3 it can be seen that the percent of decolourization of insoluble chitosan and polysaccharide retention rate are the highest.
Embodiment 3
Raw sugar in embodiment is configured to the Fructus Phyllanthi crude polysaccharides solution of 0.5 mg/ml, with Fructus Citri Limoniae acid for adjusting pH extremely
4, every 100 ml Fructus Phyllanthi polysaccharide solutions are separately added into chitosan 1.0 g, 2.0 g, 3.0 g, 4.0 g, 5.0 g, at 60 DEG C
Water-bath intermittent stirring decolours 30 min, measures and calculates percent of decolourization and polyoses content, the results are shown in Table 4 after being filtered to remove residue.
Table 4 chitosan dosage is on polysaccharide percent of decolourization and the impact of polyoses content
From table 4, it can be seen that chitosan dosage percent of decolourization increase when 2.0 g～3.0g is relatively big and polyoses content changes not
Greatly, when 3.0 g～4.0 g, percent of decolourization change is little and polyoses content changes greatly, and considering chitosan dosage should control
System is between 2.0g～3.0g (every 100 ml Fructus Phyllanthi polysaccharide, concentration is 0.5 mg/ml).
Embodiment 4
Raw sugar in embodiment is configured to the Fructus Phyllanthi crude polysaccharides solution of 0.5 mg/ml, and every 100 ml Fructus Phyllanthis are many
Sugar juice is separately added into and adjusts pH value to 3.0,4.0,5.0,6.0,7.0 with citric acid or 20% NaOH, then be separately added into shell
Polysaccharide 1.5 g, decolours 30 min at 60 DEG C of water-bath intermittent stirrings, measures and calculate percent of decolourization and polysaccharide contains after being filtered to remove residue
Amount, the results are shown in Table 5.
Table 5 PH is on polysaccharide percent of decolourization and the impact of polyoses content
As can be seen from Table 5, percent of decolourization reduces with the increase of pH, and percent of decolourization is very poor in neutral and alkaline conditions, and
PH polyoses content between 3.0～5.0 is higher.Consider decolouring pH should control between 3.0～5.0.
Embodiment 5
Raw sugar in embodiment is configured to the Fructus Phyllanthi crude polysaccharides solution of 0.5 mg/ml, with Fructus Citri Limoniae acid for adjusting pH extremely
4, in every 100 ml Fructus Phyllanthi polysaccharide solutions, difference chitosan 1.5g, is respectively placed in 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C of water
Bath intermittent stirring decolours 30 min, measures and calculates percent of decolourization and polyoses content, the results are shown in Table 6 after being filtered to remove residue.
Table 6 temperature is on polysaccharide percent of decolourization and the impact of polyoses content
As can be seen from Table 6, bleaching temperature, in time increasing to 50 DEG C for 30 DEG C, can significantly improve percent of decolourization, and 50
Change inconspicuous after DEG C.40 DEG C～the change of 50 DEG C of polyoses contents little, 50 DEG C～60 DEG C of polyoses contents change relatively
Greatly, so, bleaching temperature should be 30-50 DEG C.
Embodiment 6
Raw sugar in embodiment is configured to the Fructus Phyllanthi crude polysaccharides solution of 0.5 mg/ml, with Fructus Citri Limoniae acid for adjusting pH extremely
4, respectively chitosan 0.5g in every 100 ml Fructus Phyllanthi polysaccharide solutions, be placed in 60 DEG C, the decolouring of water-bath intermittent stirring, in 10min,
20min, 30 min, 40min, 50min, 60min are separately sampled, measure and calculate percent of decolourization and polysaccharide contains after being filtered to remove residue
Amount, the results are shown in Table 7.
Table 7 bleaching time is on polysaccharide percent of decolourization and the impact of polyoses content
| Bleaching time (min) | Percent of decolourization (%) | Polysaccharide retention rate (%) |
| 10 | 45.12 | 68.26 |
| 30 | 60.12 | 72.58 |
| 60 | 59.54 | 68.21 |
| 90 | 58.26 | 62.02 |
| 120 | 56.25 | 53.25 |
As can be seen from Table 7, percent of decolourization is increase in time and increases, and 60～90 min percent of decolourizations are constant, and many
Sugar content is increase in time and reduces, and higher at 30～60 min polysaccharide content, 60～90 min contain
Amount reduces.Consider bleaching time should control between 30 60 min.
Embodiment 7
Embodiment 7 orthogonal experiments-synthesis necessary technology
By the result of embodiment 3～6 experiment of single factor, 4 factor 3 levels of choosing carry out orthogonal experiment.Factor level sets
Meter is shown in Table 8.Data process and use synthesis necessary technology.The results are shown in Table 9
Table 8 factor level
Table 9 orthogonal table and result
For polysaccharide percent of decolourization, factor primary and secondary: A > D > C > B, optimum A2B1C2D3, A are principal elements, B
It it is secondary cause；For polysaccharide retention rate, factor primary and secondary: A > B > D > C. optimum A1B3C2D2, A be main because of
Element, C is secondary cause；Considering, with A2B3C2D2 as optimum condition, in the most every 100 ml Fructus Phyllanthi polysaccharide, shell gathers
Sugar consumption is optimum condition at 2.5 g, time 60min, bleaching temperature 40 DEG C, PH 4.Through checking, it was demonstrated that in this condition
Lower polysaccharide percent of decolourization and polysaccharide retention rate are the highest.
Optimum range: chitosan dosage 2.0～2.5 g in every 100 ml Fructus Phyllanthi polysaccharide, time 45～60 min,
Bleaching temperature should be 40-50 DEG C, and PH is 3.0～4.0.
Embodiment 8
Take the Fructus Phyllanthi polysaccharide after decolouring, add in DMEM culture medium, make final concentration of 3.2,1.6,0.8,0.4,
0.2mg/ ml.The digestion of HepG2, EC109 cell strain is made the cell suspension of 5 × 10-4/L, every hole 100 μ L's
Amount be inoculated in 96 well culture plates, with the DMEM culture fluid containing 10% hyclone and Fructus Phyllanthi polysaccharide put 37 DEG C, 5%
Cultivating 24h in CO2 cell culture incubator, after requiring to cultivate according to experiment packet, it is molten that every hole adds 20 μ L MTT (5mg/mL)
Liquid, sucking-off supernatant after continuing to hatch 4 h at 37 DEG C, every hole adds 150 μ L DMSO, shakes up with plate shaker,
At 490 nm wavelength, the absorbance (A value) in every hole is detected by microplate reader.With 5-fluorouracil (5-FU) as positive drug.Logical
The comparison crossing absorbance understands the anti-tumor activity of medicine.
The table 10 suppression ratio to hepatoma H22 cells cell
As known from Table 10: Fructus Phyllanthi polysaccharide is better than 5-FU to the inhibitory activity of hepatoma H22 cells.
The table 11 suppression ratio to esophageal cancer cell strain EC109
As known from Table 11: Fructus Phyllanthi polysaccharide is better than 5-FU to the inhibitory activity of esophageal cancer cell strain EC109.
Claims (4)
1. a preparation method for Fructus Phyllanthi polysaccharide, its step includes:
(1) Fructus Phyllanthi enucleation, with 50～80 DEG C of hot-water soaks 10～60 min, then pulls an oar, serosity is heated to reflux 1
～after 5h, hot extract filtered while hot；
(2) gained filtrate being concentrated into original volume 20～30%, being adjusted to solution ethanol content with 95% edible ethanol is 70
～85%, stand 8～15hr, centrifugal, precipitation is washed 1～3 time with appropriate 80% ethanol, dehydrated alcohol successively,
Precipitate freeze-dried Fructus Phyllanthi crude polysaccharides；
(3) Fructus Phyllanthi crude polysaccharides is made into 0.2～0.8mg/ml aqueous solution, with Fructus Citri Limoniae acid for adjusting pH to 3.0～4.0,
Bleaching temperature controls at 40～50 DEG C, and through 45～60 min, every 100 ml Fructus Phyllanthi crude polysaccharides solution add 2.0
～2.5 the chitosan of g decolour；
(4) gained filtrate is concentrated, precipitates, filters, freeze-dried Fructus Phyllanthi polysaccharide；
It is characterized in that:
Fructus Phyllanthi polysaccharide prepared by above-mentioned steps 1 ~ 4 is to hepatoma H22 cells, and esophageal cancer cell strain EC109 has and presses down
System activity is stronger than 5-FU.
2. according to method described in claim 1, it is characterised in that: in step (1), Fructus Phyllanthi is 1:5 with the solid-liquid ratio of hot water
～1:10.
3. according to method described in claim 1, it is characterised in that: in step (2), filtrate concentrates and uses normal pressure concentrate or subtract
Pressure concentrates.
4. according to method described in claim 1, it is characterised in that: in step (2) and step (4) cryogenic temperature be-4 DEG C～-
20℃。
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| CN107309438B (en) * | 2017-07-14 | 2021-03-09 | 福建农林大学 | Method for preparing nano-silver composite particles from phyllanthus emblica polysaccharide |
| CN108342518A (en) * | 2018-01-16 | 2018-07-31 | 云南省农业科学院甘蔗研究所 | A kind of emblic brown sugar and preparation method thereof |
| CN108782870A (en) * | 2018-07-17 | 2018-11-13 | 佛山推启农业研究院（普通合伙） | A kind of anti-tumor healthcare tea and preparation method thereof |
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