CN110092845A - A kind of low molecular weight Sepia polysaccharide and preparation method and application - Google Patents
A kind of low molecular weight Sepia polysaccharide and preparation method and application Download PDFInfo
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Abstract
Present disclose provides a kind of low molecular weight Sepia polysaccharide and preparation method and application, chemical structural formula is as follows:Weight average molecular weight is 1kDa~9kDa.Preparation method is that Sepia polysaccharide is carried out degradation and obtains low molecular weight Sepia polysaccharide using acid as degradation agent.The disclosure it is discovered by experiment that when by Sepia polysaccharide degrade to weight average molecular weight be 1kDa~9kDa when, compared with natural Sepia polysaccharide, have higher extracorporeal anti-tumor and immune-enhancing activity.
Description
Technical field
This disclosure relates to Sepia polysaccharide preparation technical field, and in particular to a kind of low molecular weight Sepia polysaccharide and preparation
Method and application.
Background technique
Here statement only provides background information related with the disclosure, without necessarily constituting the prior art.
Cuttlefish belongs to Mollusca, Cephalopoda, Decapoda, Sepiidae, and amount of fishing annual in recent years is more than 2 ×
106t.Many people think that Cuttlefish Ink is only the waste in cuttlefish meat products processing link.In fact, Cuttlefish Ink is as simply
Traditional Chinese medicine has the medicinal history in over thousands of year in China.To the research of Cuttlefish Ink mainly around polysaccharide therein, polypeptide, black
The ingredients such as pigment.According to known to the disclosed invention people, the black polysaccharide (SIP) of natural Sepiella maindroni (Sepiella maindroni)
Due to extremely low without sulfate or sulfate content, SIP is carried out sulphation modification by many researcher's selections, to improve its life
Object activity.
According to known to the disclosed invention people, SIP and its sulfated derivative SIP-SII are thin in Experiment on therapy human ovarian cancer
Born of the same parents SKOV-3, human mouth epidermoid carcinoma cell KB, human liver cancer cell HepG2, Human Prostate Cancer Cells PC-3, human prostata cancer are thin
Born of the same parents DU-145, human breast cancer cell MDA-MB-231, B16 mouse melanoma cell line F10 etc. show good effect,
And antitumor action can be played by number of mechanisms, including inducing apoptosis of tumour cell, inhibit tumor angiogenesis, is anti-swollen
Tumor invasion and transfer, adjust immune, reduction toxic and side, adjuvant chemotherapy drug plays a role, anti-oxidant etc. multiple
Aspect.SIP remove DPPH, OH, in terms of performance it is good, while there is good total antioxidation energy
Power (total anti-oxidation capacity, T-AOC).SIP and SIP-SII can be by improving immunocyte quantity, adjusting
The specificity and nospecific immunity of the approach enhancing body such as ganglion cell's factor generation.
However, studying by the disclosed invention people, the bioactivity of above-mentioned SIP and its sulfated derivative SIP-SII are only
It is limited to natural products purification or sulphation, and its bioactivity is lower.
Summary of the invention
In order to solve the deficiencies in the prior art, purpose of this disclosure is to provide a kind of low molecular weight Sepia polysaccharide and preparations
Method and application obtain low molecular weight Sepia polysaccharide by degradation Sepia polysaccharide, and specific low molecular weight Cuttlefish Ink is more
The bioactivity of sugar is higher than the Sepia polysaccharide naturally purified.
To achieve the goals above, the technical solution of the disclosure are as follows:
On the one hand, a kind of low molecular weight Sepia polysaccharide, chemical structural formula are as follows:
Weight average molecular weight is 1kDa~9kDa.
The disclosure it is discovered by experiment that when by Sepia polysaccharide degrade to weight average molecular weight be 5kDa~9kDa when, with day
Right Sepia polysaccharide is compared, and has higher extracorporeal anti-tumor and immune-enhancing activity.
On the other hand, a kind of preparation method of low molecular weight Sepia polysaccharide is more by Cuttlefish Ink using acid as degradation agent
Sugar carries out degradation and obtains low molecular weight Sepia polysaccharide, and the low molecular weight is the weight average molecular weight lower than 10kDa.
The disclosure be found through experiments that Sepia polysaccharide cannot by common glycosidase, as hyaluronidase, alpha-amylase,
Beta amylase, chitinase, cellulose degraded, the disclosure are for the first time degraded Sepia polysaccharide using acid.
The step of present disclose provides a kind of preparation methods of low molecular weight Sepia polysaccharide: to the water-soluble of Sepia polysaccharide
Acid is added in liquid, then heating reaction, low molecular weight Sepia polysaccharide is obtained after reaction.
The third aspect, a kind of low molecular weight Sepia polysaccharide that above-mentioned preparation method obtains.The low molecular weight Cuttlefish Ink is more
The low molecular weight Sepia polysaccharide for being 1kDa~9kDa containing weight average molecular weight in sugar can be improved the external anti-of Sepia polysaccharide
Tumour and immune-enhancing activity.
Fourth aspect, a kind of pharmaceutical composition, including above-mentioned low molecular weight Sepia polysaccharide.
5th aspect, a kind of pharmaceutical preparation, including above-mentioned low molecular weight Sepia polysaccharide or aforementioned pharmaceutical compositions and medicine
Acceptable auxiliary material and/or carrier on.
6th aspect, a kind of above-mentioned low molecular weight Sepia polysaccharide, aforementioned pharmaceutical compositions or said medicine preparation are being made
Application in standby anti-tumor drug and/or enhancing immune drug.
The disclosure has the beneficial effect that
1. disclosure first passage acid degrades Sepia polysaccharide.
2. the disclosure is experimentally confirmed, after Sepia polysaccharide is degraded, low molecular weight cuttlefish that only extent of polymerization reduces
Black polysaccharide can be improved the anti-tumor activity of Sepia polysaccharide and the facilitation to spleen lymphocyte proliferation.The low molecule
The low molecular weight Sepia polysaccharide for being 1kDa~9kDa containing molecular weight in Sepia polysaccharide is measured, especially with the work of 5kDa~7kDa
Property is best.
Detailed description of the invention
The Figure of description for constituting a part of this disclosure is used to provide further understanding of the disclosure, and the disclosure is shown
Meaning property embodiment and its explanation do not constitute the improper restriction to the disclosure for explaining the disclosure.
Fig. 1 is the infrared spectrogram of SIP in embodiment；
Fig. 2 is the infrared spectrogram of LMWSIP-1 prepared by embodiment 18；
Fig. 3 is the infrared spectrogram of LMWSIP-2 prepared by embodiment 18；
Fig. 4 is the infrared spectrogram of LMWSIP-3 prepared by embodiment 18；
Fig. 5 is histogram of the sample to the HepG2 inhibiting effect being proliferated of the preparation of embodiment 18；
When Fig. 6 is 100 μ g/mL of sample prepared by embodiment 18, the histogram of the apoptosis rate of HepG2；
When Fig. 7 is 500 μ g/mL of sample prepared by embodiment 18, the histogram of the apoptosis rate of HepG2；
To the histogram of the inhibiting rate of HepG2 migration when Fig. 8 is the 4 μ g/mL of sample of the preparation of embodiment 18；
To the histogram of the inhibiting rate of HepG2 migration when Fig. 9 is the 20 μ g/mL of sample of the preparation of embodiment 18；
Figure 10 is the histogram that sample prepared by embodiment 18 influences kunming mice spleen lymphocyte proliferation；
Figure 11 is the histogram that the kunming mice spleen lymphocyte proliferation of sample ConA induction prepared by embodiment 18 influences.
Specific embodiment
It is noted that described further below be all exemplary, it is intended to provide further instruction to the disclosure.Unless another
It indicates, all technical and scientific terms used herein has usual with disclosure person of an ordinary skill in the technical field
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root
According to the illustrative embodiments of the disclosure.As used herein, unless the context clearly indicates otherwise, otherwise singular
Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation, device, component and/or their combination.
The present disclosure proposes a kind of low molecular weight Sepia polysaccharide and preparation method and applications, pass through Sepia polysaccharide of degrading
Low molecular weight Sepia polysaccharide is obtained, and the bioactivity of specific low molecular weight Sepia polysaccharide is higher than the cuttlefish naturally purified
Black polysaccharide.
A kind of exemplary embodiment of the disclosure provides a kind of low molecular weight Sepia polysaccharide (LMWSIPs), chemistry
Structural formula is as follows:
Weight average molecular weight is 1kDa~9kDa.
The disclosure it is discovered by experiment that when by Sepia polysaccharide degrade to weight average molecular weight be 5kDa~9kDa when, with day
Right Sepia polysaccharide is compared, and has higher extracorporeal anti-tumor and immune-enhancing activity.
In one or more embodiments of the embodiment, weight average molecular weight is 5kDa~7kDa.By it is experimentally confirmed that should
The extracorporeal anti-tumor and immune-enhancing activity of low molecular weight Sepia polysaccharide under molecular weight are more excellent.
The another embodiment of the disclosure provides a kind of preparation method of low molecular weight Sepia polysaccharide, utilizes acid
As degradation agent, Sepia polysaccharide is subjected to degradation and obtains low molecular weight Sepia polysaccharide, the low molecular weight is lower than 10kDa
Weight average molecular weight.
The disclosure be found through experiments that Sepia polysaccharide cannot by common glycosidase, as hyaluronidase, alpha-amylase,
Beta amylase, chitinase, cellulose degraded, the disclosure are for the first time degraded Sepia polysaccharide using acid.
Acid described in the disclosure be chemical field general significance acid, such as hydrochloric acid, phosphoric acid etc., the one of the embodiment
In kind or various embodiments, the acid is trifluoroacetic acid.It since trifluoroacetic acid boiling point is lower, easily removes, thus selects trifluoro
Acetic acid is degraded.
In one or more embodiments of the embodiment, trifluoro second step: is added into the aqueous solution of Sepia polysaccharide
Acid, then heating reaction, obtains low molecular weight Sepia polysaccharide after reaction.
In the series embodiment, reaction temperature is 60~100 DEG C.It can guarantee the degradation speed of Sepia polysaccharide at this temperature
Rate is very fast.Reaction time is 1~8h.After the reaction time, it can guarantee that Sepia polysaccharide degradation is complete.
The disclosure is found through experiments that, the excessively high primary structure that can seriously destroy Sepia polysaccharide of acid concentration, especially when
When acid concentration is not less than 1mol/L, the product after Sepia polysaccharide degradation is mostly oligosaccharides or monosaccharide, and low point of 5kDa~9kDa
Son amount Sepia polysaccharide is lower.In order to promoted 5kDa~9kDa low molecular weight Sepia polysaccharide yield, the embodiment
In one or more embodiments, sour concentration is 0.4~0.6mol/L.At this point, reaction temperature is 75~85 DEG C, the reaction time
3.5~4h can guarantee Sepia polysaccharide fully reacting, while the reaction condition temperature and time is moderate.
In order to obtain the low molecular weight Sepia polysaccharide of more specified molecular weight, one or more implementations of the embodiment
In example, the material after reaction is isolated and purified, described the step of isolating and purifying are as follows: by the dry materials after reaction, then will
Material after drying adds water to filter, and filtered liquid is then carried out gel filtration chromatography separation.
Specific steps are as follows:
(1) material after reaction is dried；
(2) water that 1 volume is added in the material after drying is dissolved, after filtering, using 150 tomographic system of AKTA avant
Connection Sephadex G-50 gel column (1.2 × 100cm) is isolated and purified, and every pipe collects 2 volume eluents automatically；
(3) step (2) the 2nd~8 pipe eluent is collected to be freeze-dried.
The low molecular weight Sepia polysaccharide that molecular weight is 5kDa~9kDa can be obtained.
When being freeze-dried the 5th~8 pipe eluent of collection in step (3), can obtain weight average molecular weight is
The low molecular weight Sepia polysaccharide of 5kDa~7kDa.
A kind of the third low molecular weight Cuttlefish Ink obtained embodiment there is provided above-mentioned preparation method of the disclosure is more
Sugar.The low molecular weight Sepia polysaccharide for being 5kDa~9kDa containing weight average molecular weight in the low molecular weight Sepia polysaccharide, can
Improve the extracorporeal anti-tumor and immune-enhancing activity of Sepia polysaccharide.
Embodiment there is provided a kind of pharmaceutical composition, including above-mentioned low molecular weight Cuttlefish Ink are more for the 4th kind of the disclosure
Sugar.The composition can choose the modes such as oral, rectally, local administration, parenterai administration and be administered.
Embodiment there is provided a kind of pharmaceutical preparations, including above-mentioned low molecular weight Sepia polysaccharide for the 5th kind of the disclosure
Or aforementioned pharmaceutical compositions and pharmaceutically acceptable auxiliary material and/or carrier.Auxiliary material and/or carrier can choose: ion exchange
Agent, aluminium oxide, aluminum stearate, lecithin, haemocyanin, phosphate, glycerol, sorb ester, potassium sorbate, saturation plant fat
Acid, water, salt, electrolyte etc..
Embodiment there is provided a kind of above-mentioned low molecular weight Sepia polysaccharide, said medicines to combine for the 6th kind of the disclosure
Object or said medicine preparation are preparing anti-tumor drug and/or are enhancing the application in immune drug.
Molecular weight described in the disclosure is weight average molecular weight.
In order to enable those skilled in the art can clearly understand the technical solution of the disclosure, below with reference to tool
The technical solution of the disclosure is described in detail in the embodiment of body.
The preparation method that Sepia polysaccharide (SIP) is used in the embodiment of the present disclosure, with reference to Liu CH, Li XD, Li YH, et
al.Structural characterisation and antimutagenic activity of a novel
polysaccharide isolated from Sepiella maindroni ink.Food Chem,2008,110(4):
807-813。
By characterization, molecular structural formula isWeight
Average molecular weight is 11.3kDa.
Embodiment 1
It is degraded using hyaluronidase to SIP.
Degradation condition are as follows: SIP concentration 10mg/mL, the enzyme activity of hyaluronidase are 50U/mg, and degradation pH is 7, degradation temperature
It is 37 DEG C, Tris-HCl buffer concentration 50mmol/L, degradation time is for 24 hours.
Embodiment 2
It is degraded using chondrosulphatase to SIP.
Degradation condition are as follows: SIP concentration 10mg/mL, the enzyme activity of chondroitin sulfate are 30U/mg, and degradation pH is 7, degradation temperature
It is 30 DEG C, Tris-HCl buffer concentration 50mmol/L, degradation time is for 24 hours.
Embodiment 3
It is degraded using Heparinase I to SIP.
Degradation condition are as follows: SIP concentration 10mg/mL, the enzyme activity of Heparinase I are 60U/mg, and degradation pH is 7.5, degradation temperature
It is 30 DEG C, Tris-HCl buffer concentration 50mmol/L, degradation time is for 24 hours.
Embodiment 4
It is degraded using Heparinase I I to SIP.
Degradation condition are as follows: the enzyme activity of SIP concentration 10mg/mL, Heparinase I I are 20U/mg, and degradation pH is 7.5, degradation temperature
It is 30 DEG C, Tris-HCl buffer concentration 50mmol/L, degradation time is for 24 hours.
Embodiment 5
It is degraded using Heparinase I II to SIP.
Degradation condition are as follows: the enzyme activity of SIP concentration 10mg/mL, Heparinase I II are 35U/mg, and degradation pH is 7.5, degradation temperature
Degree is 30 DEG C, Tris-HCl buffer concentration 50mmol/L, and degradation time is for 24 hours.
Embodiment 6
It is degraded using alpha-amylase to SIP.
Degradation condition are as follows: SIP concentration 10mg/mL, the enzyme activity of alpha-amylase are 1000U/mg, and degradation pH is 6.9, degradation temperature
Degree is 20 DEG C, PBS buffer concentration 20mmol/L, and degradation time is for 24 hours.
Embodiment 7
It is degraded using beta amylase to SIP.
Degradation condition are as follows: SIP concentration 10mg/mL, the enzyme activity of beta amylase are 100U/mg, and degradation pH is 6, degradation temperature
It is 50 DEG C, PBS buffer concentration 20mmol/L, degradation time is for 24 hours.
Embodiment 8
It is degraded using chitinase to SIP.
Degradation condition are as follows: SIP concentration 10mg/mL, the enzyme activity of chitinase are 2U/mg, and degradation pH is 6, and degradation temperature is
37 DEG C, NaAc_HAc buffer solution concentration 10mmol/L, degradation time is for 24 hours.
Embodiment 9
It is degraded using cellulase to SIP.
Degradation condition are as follows: SIP concentration 10mg/mL, the enzyme activity of cellulase are 3U/mg, and degradation pH is 4.8, degradation temperature
It is 50 DEG C, citric acid-sodium citrate buffer concentration 20mmol/L, degradation time is for 24 hours.
Embodiment 10
It is degraded using hemicellulase to SIP.
Degradation condition are as follows: SIP concentration 10mg/mL, the enzyme activity of hemicellulase are 1.5U/mg, and degradation pH is 4.8, degradation
Temperature is 50 DEG C, citric acid-sodium citrate buffer concentration 20mmol/L, and degradation time is for 24 hours.
Utilize OD232nmThe quantity of unsaturated double-bond, determines Examples 1 to 5 in the absorbance value variation detection substrate at place
Degree of Enzymatic Hydrolysis, however OD232nmDo not increase.Change the Degree of Enzymatic Hydrolysis for determining embodiment 6~10, however chromatography using chromatographic peak quantity
Peak number amount is 1.Showing Examples 1 to 10 can not degrade SIP.
It is degraded using trifluoroacetic acid to SIP.
Embodiment 11
SIP 10mg is weighed, is dissolved in the TFA solution of 5mL 1.0mol/L, is placed in heating and condensation reflux unit and carries out
Degradation reaction, temperature are 100 DEG C, heating time 8h.In degradation process, appropriate catabolite is taken to carry out every 0.5h
HPTLC analysis (solvent is normal propyl alcohol: water: triethylamine=50:30:0.7, and color developing agent is anisaldehyde dyeing liquor), after reacting 8h,
Pass through rotary evaporation dry reaction product immediately, it is ensured that the TFA in reaction system is completely removed.In addition, pass through HPTLC as a result,
Carefully compare the degradation situation of SIP under each degradation condition.
Embodiment 12
SIP 10mg is weighed, is dissolved in the TFA solution of 5mL 1.0mol/L, is placed in heating and condensation reflux unit and carries out
Degradation reaction, temperature are 80 DEG C, heating time 8h.In degradation process, appropriate catabolite is taken to carry out HPTLC every 0.5h
Analysis (solvent is normal propyl alcohol: water: triethylamine=50:30:0.7, and color developing agent is anisaldehyde dyeing liquor), after reacting 8h, immediately
Pass through rotary evaporation dry reaction product, it is ensured that the TFA in reaction system is completely removed.In addition, passing through HPTLC as a result, carefully
Compare the degradation situation of SIP under each degradation condition.
Embodiment 13
SIP 10mg is weighed, is dissolved in the TFA solution of 5mL 1.0mol/L, is placed in heating and condensation reflux unit and carries out
Degradation reaction, temperature are 60 DEG C, heating time 8h.In degradation process, appropriate catabolite is taken to carry out HPTLC every 0.5h
Analysis (solvent is normal propyl alcohol: water: triethylamine=50:30:0.7, and color developing agent is anisaldehyde dyeing liquor), after reacting 8h, immediately
Pass through rotary evaporation dry reaction product, it is ensured that the TFA in reaction system is completely removed.In addition, passing through HPTLC as a result, carefully
Compare the degradation situation of SIP under each degradation condition.
Embodiment 14
SIP 10mg is weighed, is dissolved in the TFA solution of 5mL 0.5mol/L, is placed in heating and condensation reflux unit and carries out
Degradation reaction, temperature are 100 DEG C, heating time 8h.In degradation process, appropriate catabolite is taken to carry out every 0.5h
HPTLC analysis (solvent is normal propyl alcohol: water: triethylamine=50:30:0.7, and color developing agent is anisaldehyde dyeing liquor), after reacting 8h,
Pass through rotary evaporation dry reaction product immediately, it is ensured that the TFA in reaction system is completely removed.In addition, pass through HPTLC as a result,
Carefully compare the degradation situation of SIP under each degradation condition.
Embodiment 15
SIP 10mg is weighed, is dissolved in the TFA solution of 5mL 0.5mol/L, is placed in heating and condensation reflux unit and carries out
Degradation reaction, temperature are 80 DEG C, heating time 8h.In degradation process, appropriate catabolite is taken to carry out HPTLC every 0.5h
Analysis (solvent is normal propyl alcohol: water: triethylamine=50:30:0.7, and color developing agent is anisaldehyde dyeing liquor), after reacting 8h, immediately
Pass through rotary evaporation dry reaction product, it is ensured that the TFA in reaction system is completely removed.In addition, passing through HPTLC as a result, carefully
Compare the degradation situation of SIP under each degradation condition.
Embodiment 16
SIP 10mg is weighed, is dissolved in the TFA solution of 5mL 0.5mol/L, is placed in heating and condensation reflux unit and carries out
Degradation reaction, temperature are 60 DEG C, heating time 8h.In degradation process, appropriate catabolite is taken to carry out HPTLC every 0.5h
Analysis (solvent is normal propyl alcohol: water: triethylamine=50:30:0.7, and color developing agent is anisaldehyde dyeing liquor), after reacting 8h, immediately
Pass through rotary evaporation dry reaction product, it is ensured that the TFA in reaction system is completely removed.In addition, passing through HPTLC as a result, carefully
Compare the degradation situation of SIP under each degradation condition.
Embodiment 17
SIP 10mg is weighed, is dissolved in the TFA solution of 5mL 0.1mol/L, is placed in heating and condensation reflux unit and carries out
Degradation reaction, temperature are 100 DEG C, heating time 8h.In degradation process, appropriate catabolite is taken to carry out every 0.5h
HPTLC analysis (solvent is normal propyl alcohol: water: triethylamine=50:30:0.7, and color developing agent is anisaldehyde dyeing liquor), after reacting 8h,
Pass through rotary evaporation dry reaction product immediately, it is ensured that the TFA in reaction system is completely removed.In addition, pass through HPTLC as a result,
Carefully compare the degradation situation of SIP under each degradation condition.
The complete reaction used time under 11~17 acidolysis condition of embodiment is as shown in table 1.
The complete reaction used time under 1 embodiment of table, 11~17 acidolysis condition
It selects Biogel P-2 gel chromatography column to isolate and purify the product of embodiment 11 and embodiment 12, is divided
Sub- scale sign, molecular weight ranges occupy sizable ratio in the product of 100~1800Da.Obviously, this Partial digestion product
The oligosaccharides or monosaccharide obtained for degradation, it was demonstrated that TFA excessive concentration used by condition (1) and (2) can not be used to prepare
LMWSIPs。
Sephadex G-50 gel chromatography column is selected to isolate and purify the product of embodiment 14 and embodiment 15, into
Row molecular weight characterization, under conditions of TFA concentration is 0.5mol/L, 100 DEG C of heating temperature will cause catabolite molecular weight point
Cloth is uneven, and it is too low to show as middle-molecular-weihydroxyethyl catabolite proportion, and low molecular weight catabolite proportion is excessively high.?
Under conditions of TFA concentration is 0.5mol/L, when heating temperature is reduced to 80 DEG C, the molecular weight point of catabolite can be obviously improved
Cloth.In addition, reaction temperature is reduced to 80 DEG C by 100 DEG C, required time is only increased to by 3h from the point of view of the time required to reaction
3.5h belongs in tolerance interval.Condition after thus selection example 15 optimizes continues to prepare LMWSIPs, such as embodiment 18.
Embodiment 18
SIP 10mg is weighed, is dissolved in the TFA solution that 5mL concentration is 0.5mol/L, in 80 DEG C of reaction 3.5h, rotates immediately
TFA in evaporative removal reaction system, makes reaction terminating.Degassing distilled water 1mL is added into the reaction product after being evaporated, uses
After 0.22 μm of membrane filtration, Sephadex G-50 gel column (1.2 × 100cm) is connected through 150 tomographic system of AKTA avant
It isolates and purifies, sets every pipe and collect eluent 2mL automatically, obtain 26 pipes altogether.Mobile phase is degassing distilled water, flow velocity 0.5mL/
min.Each pipe eluent is placed in vial, freeze-drying obtains LMWSIPs.
Molecular weight characterization is carried out to 26 pipe LMWSIPs, the results are shown in Table 2.
The sample cell number of 2 different molecular weight distribution of table
Since the molecular weight of undegradable SIP is 11.3kDa, molecular weight is more than the low-molecular-weight polysaccharide sample size of 9kDa
It is less, and molecular weight and preceding difference of degrading are little；And work as the mixture that the too low catabolite of molecular weight may be monosaccharide and oligosaccharides,
Thus give up the decomposition product that molecular weight is less than 3kDa greater than 9kDa and molecular weight.
Using range of molecular weight distributions for 7kDa~9kDa sample as LMWSIP-1, be 5kDa with range of molecular weight distributions
The sample of~7kDa as LMWSIP-2, using range of molecular weight distributions for 3kDa~5kDa sample as LMWSIP-3, continue
It is characterized.
The infrared spectroscopy of SIP, LMWSIP-1, LMWSIP-2, LMWSIP-3 as shown in figures 1-4, show SIP, LMWSIP-
1, the infared spectrum of LMWSIP-2 is almost the same, and the infared spectrum of LMWSIP-3 and other 3 samples has significant difference, especially
It is in fingerprint region.Illustrate LMWSIP-1, LMWSIP-2 compared with SIP, there is no the variation that functional group's type occurs, i.e. acidolysis
When effect proceeds to the two degree, the structure of each monosaccharide is not destroyed, and is only had occurred under polysaccharide sugar chain extent of polymerization
Drop；And when polysaccharide is degraded between molecular weight area locating for LMWSIP-3, thus it is speculated that its primary structure is destroyed, the knot of each monosaccharide
Structure is even also destroyed, and dissociate more polar groups, such as carboxyl, hydroxyl.
Characterization of biological activity is carried out to SIP, LMWSIP-1, LMWSIP-2, LMWSIP-3.
1LMWSIPs and SIP extracorporeal anti-tumor function characterization.
1.1 influence using mtt assay detection LMWSIPs and SIP to tumor cell proliferation.
Cell inoculation
The HepG2 cell of logarithmic growth phase.It is consistent in digestion and collection step and cell passage operation.It will collect
Cell be resuspended with the complete high glucose medium 1mL of DMEM.10 μ L of re-suspension liquid is drawn, is diluted with 10 times of volume PBS, is carried out thin
Born of the same parents count.By cell counts, it is 5.0 that the complete high glucose medium of DMEM of re-suspension liquid certain volume, which is diluted to density,
×104cells/mL.Cell suspension after dilution is mixed, is inoculated on 96 well culture plates with the volume of every 100 μ L of hole.This
Outside, blank control group, negative control group, positive controls, experimental group need to be arranged in this experiment.It is not inoculated in blank control wells thin
The complete 100 μ L of high glucose medium of DMEM is only added in born of the same parents.Remaining 3 groups are accessed cell according to the method.Then, to containing culture medium
100 μ L of PBS is respectively added in the periphery adjacent holes in hole and avoids edge effect to maintain moisture constant in culture plate.Finally, by 96
Well culture plate is in 37 DEG C, 5%CO2, saturated humidity condition constant incubator in cultivate for 24 hours.
Agent-feeding treatment
Agent-feeding treatment pays attention to distinguishing blank control group, negative control group, positive controls, experimental group.For blank control
Group: the complete 100 μ L of high glucose medium of DMEM is continuously added into each corresponding aperture, and 5 multiple holes are set；For negative control group:
The complete 100 μ L of high glucose medium of DMEM is added to each corresponding aperture, and 5 multiple holes are set；For positive controls: to each corresponding aperture
The 100 μ L of 5-FU solution of final concentration of 20 μ g/mL is added, and 5 multiple holes are set；For experimental group: take certain volume SIP,
LMWSIP-1, LMWSIP-2, LMWSIP-3 mother liquor, respectively dilute certain multiple, and 100 μ L are added to each corresponding aperture, keep each drug whole
Concentration is respectively 500,100,20,4,0.8 μ g/mL, and 5 multiple holes are arranged in each concentration.Culture plate is placed in the training of constant temperature cell
It supports in case and cultivates 48h.
Testing result
96 well culture plates are taken out, every hole is added the 15 μ L of MTT solution of 5mg/mL, puts back to and continue to cultivate in cell incubator.
After 4h, culture plate is taken out, 10min is centrifuged in 3000r/min, is carefully inhaled with syringe and abandon supernatant, be careful not to encounter hole
Bottom.150 μ L of DMSO is added to every hole, then measures each hole absorbance value with microplate reader.Microplate reader parameter: wavelength selection
490nm shakes plate 5min before every plate test with middling speed.Each hole absorbance value is recorded, calculates cell inhibitory rate according to following formula:
Statistical analysis
Using SPSS Statistics software, one-way anova inspection carried out to each group proliferation rate, when p < 0.05 recognizes
For with significant difference, when p < 0.01, thinks there is extremely significant sex differernce.
Influence of the 1.2 Flow cytometry LMWSIPs and SIP to apoptosis of tumor cells
Cell inoculation
The HepG2 cell of logarithmic growth phase.It is consistent in digestion and collection step and cell passage operation.It will collect
Cell be resuspended with the complete high glucose medium 1mL of DMEM.10 μ L of re-suspension liquid is drawn, is diluted with 10 times of volume PBS, is carried out thin
Born of the same parents count.By cell counts, it is 1.0 that the complete high glucose medium of DMEM of re-suspension liquid certain volume, which is diluted to density,
×105cells/mL.Cell suspension after dilution is mixed, is inoculated on 6 well culture plates with the volume of every hole 2mL.6 holes are trained
Feeding plate is placed in constant temperature cell incubator and cultivates for 24 hours.
Agent-feeding treatment
Agent-feeding treatment caution area divides negative control group, positive controls, experimental group.Before each hole dosing, careful inhale abandons old training
Support base.For negative control group: being added without medical fluid, the complete high glucose medium 2mL of DMEM is added to each corresponding aperture, and be arranged 3
Multiple holes；For positive controls: the 5-FU solution 2mL of final concentration of 20 μ g/mL being added to each corresponding aperture, and 3 multiple holes are arranged；
For experimental group: 2mL medical fluid is added to each corresponding aperture, divides equally the final concentration of SIP, LMWSIP-1, LMWSIP-2, LMWSIP-3
Not Wei 100,500 μ g/mL, and be arranged 3 multiple holes.
Influence of the Annexin V-FITC/PI combination flow cytomery drug to HepG2 apoptosis
1. drawing the old culture medium in each hole, respectively it is transferred in 15mL centrifuge tube.PBS 1mL is added to each hole, washes away remnants
Culture medium is transferred in former centrifuge tube, and is repeated the operating procedure 1 time.Trypsase-EDTA digestive juice 1mL is added to each hole,
After digesting 3min, the complete high glucose medium 3mL of DMEM is added and terminates digestion, careful piping and druming at single cell suspension, be transferred to it is former from
In heart pipe.2000r/min is centrifuged 5min, obtains cell precipitation.
2. with PBS be resuspended, washing cell precipitation, 2000r/min be centrifuged 5min, repetitive operation 2 times.
3. cell is resuspended in 1 × Binding Buffer that certain volume is added, adjustment cell concentration is 1 × 106cells/
mL。
4. each 5 μ L of Annexin V-FITC and PI is added into cell suspension, after careful mixing, it is placed in room under the conditions of being protected from light
Temperature is incubated for 20min.
5. after cell screen clothes filtration cell suspension, in 1h use stream type cell analyzer detect Apoptosis situation.
Statistical analysis
Using SPSS Statistics software, one-way anova inspection carried out to each group apoptosis rate, when p < 0.05 recognizes
For with significant difference, when p < 0.01, thinks there is extremely significant sex differernce.
1.3 scarifications detect influence of the LMWSIPs and SIP to tumor cell migration
Cell inoculation
The HepG2 cell of logarithmic growth phase.It is consistent in digestion and collection step and cell passage operation.Use DMEM
Cell density is adjusted to 2.0 × 10 by complete high glucose medium5Cells/mL is uniformly inoculated on 6 well culture plates, every pore volume
For 2mL.
Scratching
Microscopically observation cell state and fusion rate.When cell state is good and fusion rate is about 95%, 200 are used
μ L pipette tips equidistantly mark 3 scratches along ruler in each hole, and the spacing of 3 scratches is 0.5cm or more.Culture is carefully cleaned with PBS
Plate 3 times, sufficiently to remove the cell that pipette tips are crossed out.
Agent-feeding treatment
Agent-feeding treatment caution area divides negative control group, experimental group.For negative control group: medical fluid is added without, to each correspondence
Serum-free DMEM high glucose medium 2mL is added in hole, and 3 multiple holes are arranged；For experimental group: 2mL dissolution is added to each corresponding aperture
In the medical fluid of serum-free DMEM high glucose medium, the final concentration of SIP, LMWSIP-1, LMWSIP-2, LMWSIP-3 is set to be respectively
4,20 μ g/mL, and 3 multiple holes are set.After dosing, tissue culture plate is placed in cell incubator and is cultivated for 24 hours.
Cell is taken pictures
Respectively at 0,12, observe the migration situation of each hole cell for 24 hours, clapped under the brightfield mode of inverted fluorescence microscope
According to.Note that the picture-taking position of each time point should be consistent.
Interpretation of result
The area of scored area is calculated using ImageJ software, and according to following formula computation migration rate:
Statistical analysis
Using SPSS Statistics software, one-way anova inspection carried out to each group mobility, when p < 0.05 recognizes
For with significant difference, when p < 0.01, thinks there is extremely significant sex differernce.
As shown in figure 5, concentration dependent is presented to the HepG2 inhibiting effect being proliferated in LMWSIPs and SIP.0.8,4,
Under 20 μ g/mL concentration, each drug is not significantly different the HepG2 inhibiting rate being proliferated.Under 100 μ g/mL concentration, each drug pair
HepG2 proliferation inhibiting rate be more than 14%, wherein LMWSIP-2 effect is best, and inhibiting rate reaches 23%, be significantly higher than SIP,
LMWSIP-1,LMWSIP-3.Under 500 μ g/mL concentration, each drug is more than 24% to the inhibiting rate that HepG2 is proliferated, wherein
LMWSIP-2 effect is best, and inhibiting rate reaches 35%, is significantly higher than SIP, LMWSIP-1, LMWSIP-3.
It will be appreciated from fig. 6 that the apoptosis rate of HepG2 is about 3%~5% under the action of 100 μ g/mL LMWSIPs and SIP,
SIP is unobvious to the inducing effect of HepG2 apoptosis before and after degradation.
As shown in Figure 7, under 500 each drug effects of μ g/mL, the apoptosis rate of HepG2 more than 12%, is all remarkably higher than yin
Property control group；The HepG2 apoptosis rate of LMWSIP-2 induction is more than 20%, is significantly higher than SIP, LMWSIP-1, LMWSIP-3.
As shown in Figure 8, each drug acts on 12h in 4 μ g/mL concentration versus cells, the HepG2 migration after LMWSIP-2 effect
Rate is minimum, about 1.3%, substantially less than control group and other drugs group.When 4 μ g/mL acute drugs are applied to for 24 hours, LMWSIP-
1, the mobility that LMWSIP-2 acts on lower cell respectively may be about 9.8%, 2%, substantially less than control group and other drugs group.
As shown in Figure 9, each drug acts on 12h in 20 μ g/mL concentration versus cells, and SIP, LMWSIP-2, LMWSIP-3 make
HepG2 mobility after respectively may be about 2.5%, 1%, 2.4%, substantially less than control group.20 μ g/mL acute drugs are applied to
When for 24 hours, the HepG2 mobility after SIP, LMWSIP-1, LMWSIP-2, LMWSIP-3 effect respectively may be about 4.7%, 7.3%,
1.7%, 4.6%, it is significantly reduced compared with control group, and the mobility of the lower cell of LMWSIP-2 effect is substantially less than other drugs
Group.
2LMWSIPs and SIP ion vitro immunization adjustment effect characterization
2.1CCK-8 method detects influence of the LMWSIPs and SIP to mice spleen lymphocytes proliferation
(1) kunming mice is put to death using cervical dislocation, 1min is impregnated in 75% alcohol, is transferred to sterile behaviour immediately after
Make in platform.The left abdomen of mouse is cut off, spleen is taken out, is put into Hank ' the s liquid containing 5%FBS (inactivation), rejects fat and knot
Form tissue.
(2) spleen is transferred in 200 mesh cell screen clothes, is lightly ground spleen with syringe needle core, and not with Hank ' s liquid
It is disconnected to rinse, filter lapping liquid to other 1 in the aseptic culture dish for filling Hank ' s liquid through cell screen clothes.
(3) cell suspension is transferred in sterile centrifugation tube, 2000r/min is centrifuged 3min.After, careful inhale abandons supernatant
Erythrocyte cracked liquid 10mL is added into centrifuge tube for liquid, and cell is resuspended, stands 5min at room temperature.
(4) cell suspension after erythrocyte splitting is centrifuged 3min in 2000r/min, careful inhale abandons supernatant.It is added one
Determine volume 1640 complete medium of inactivation RPMI be resuspended cell, by cell count by the concentration dilution of suspension be 1.0 ×
107cells/mL。
(5) cell inoculation and dosing.Blank control group, negative control group, positive controls, experimental group are set.For sky
White control group: not inoculating cell is only added inactivation 1640 complete medium of RPMI, 200 μ L, and 5 multiple holes is arranged；For feminine gender
Control group: being added 100 μ L of cell suspension, inactivation 1640 complete medium of RPMI, 100 μ L is added, and 5 multiple holes are arranged；For
Positive controls: being added 100 μ L of cell suspension, the 100 μ L of ConA solution of final concentration of 5 μ g/mL is added, and 5 multiple holes are arranged；
For experimental group: 100 μ L of cell suspension is added, takes SIP, LMWSIP-1, LMWSIP-2, LMWSIP-3 mother liquor of certain volume,
Each dilution certain multiple, is added 100 μ L to each corresponding aperture, and making each drug final concentration is respectively 12.5,25,50,100,200 μ
5 multiple holes are arranged in g/mL, each concentration.Culture plate is placed in constant temperature cell incubator and cultivates 48h.
(6) after cultivating, 20 μ L of CCK-8 solution is added to each hole, is careful not to generate bubble, is placed in cell incubator
Middle incubation 3h.
(7) OD is measured using microplate reader450nm, and test result is substituted into following formula, it is thin that mice spleen lymph is calculated
Born of the same parents' proliferation rate.
2.2CCK-8 method detects influence of the LMWSIPs and SIP to the ConA mice spleen lymphocytes proliferation induced
Be with 2.1 differences: blank control group, negative control group, positive controls, experimental group is arranged in agent-feeding treatment.It is right
In blank control group: not inoculating cell is only added inactivation 1640 complete medium of RPMI, 200 μ L, and 5 multiple holes is arranged；For
Negative control group: being added 100 μ L of cell suspension, inactivation 1640 complete medium of RPMI, 100 μ L is added, and 5 multiple holes are arranged；
For positive controls: 100 μ L of cell suspension is added, the 100 μ L of ConA solution of final concentration of 5 μ g/mL is added, and be arranged 5
Multiple holes；For experimental group: 100 μ L of cell suspension is first added, then is separately added into ConA solution and polysaccharide solution, makes ConA in every hole
Final concentration of 5 μ g/mL, polysaccharide final concentration be respectively 12.5,25,50,100,200 μ g/mL, and each concentration be arranged 5
Multiple holes.Culture plate is placed in constant temperature cell incubator and cultivates 48h.
As shown in Figure 10, compared with negative control group, under each concentration conditions, SIP, LMWSIP-1, LMWSIP-2 are to mouse
Spleen lymphocyte proliferation has significant facilitation, and LMWSIP-3 can only be shown at maximum concentration (200 μ g/mL)
Write the proliferation for promoting splenic lymphocytes.Under 12.5,50,100 μ g/mL concentration, LMWSIP-2 compares SIP and increases to splenic lymphocytes
The facilitation grown is significantly higher than SIP.Under 12.5,25 μ g/mL concentration, LMWSIP-1 makees the promotion of spleen lymphocyte proliferation
With substantially less than SIP.LMWSIP-3 is substantially less than SIP to the facilitation of spleen lymphocyte proliferation under each concentration conditions.It should
The experimental results showed that LMWSIP-2 is in 4 groups of drugs of SIP degradation front and back to the strongest medicine of immune function of mice stimulation
Object, and instead the immunological enhancement of LMWSIP-1, LMWSIP-3 under some concentration is not so good as SIP.
As shown in Figure 11, at various concentrations, SIP cannot play synergistic function with ConA.At various concentrations, positive
Control group and medicine group can remarkably promote the proliferation of splenic lymphocytes, but the spleen lymph that SIP induces ConA before and after degradation
The facilitation of cell Proliferation is not significantly different.Under 12.5,25,50 μ g/mL concentration, while ConA and LMWSIPs is applied,
It compares and ConA is used alone, do not make significant difference to lymphocytic proliferation rate, show under middle low concentration, LMWSIPs cannot be with ConA
Play synergistic function.When sample concentration is improved to 100 μ g/mL, what LMWSIP-1, LMWSIP-2 and ConA were applied simultaneously
Significant effect, which is better than, is used alone ConA；When sample concentration is improved to 200 μ g/mL, LMWSIP-1, LMWSIP-2, LMWSIP-3
It is better than with the significant effect that ConA is applied simultaneously and ConA is used alone.The above result shows that SIP cannot be played with ConA cooperates with increasing
Effect effect, catabolite can but play the effect, but need in higher concentration condition (i.e. when concentration is not less than 100 μ g/
ML) lower section can be achieved.
SIP anti-tumour cell proliferative, induction tumour before and after degradation are determined by mtt assay, flow cytometry, scarification
Apoptosis, the effect for inhibiting tumor cell migration, the results showed that, LMWSIP-2 resists compared to SIP and remaining group catabolite
Tumor promotion is most strong.Mice spleen lymphocytes proliferation the experimental results showed that, it is thin that SIP can remarkably promote spleen lymph before and after degradation
Born of the same parents' proliferation, and LMWSIP-2, compared to SIP and remaining group catabolite, facilitation effect is more significant；Under each concentration conditions, SIP exists
Degradation front and back is not significantly different the facilitation of the spleen lymphocyte proliferation of ConA induction；Under a high concentration condition,
LMWSIPs can play synergistic function with ConA.
The foregoing is merely preferred embodiment of the present disclosure, are not limited to the disclosure, for the skill of this field
For art personnel, the disclosure can have various modifications and variations.It is all within the spirit and principle of the disclosure, it is made any to repair
Change, equivalent replacement, improvement etc., should be included within the protection scope of the disclosure.
Claims (10)
1. a kind of low molecular weight Sepia polysaccharide, characterized in that chemical structural formula is as follows:
Weight average molecular weight is 1kDa~9kDa；
Preferably, weight average molecular weight is 5kDa~7kDa.
2. a kind of preparation method of low molecular weight Sepia polysaccharide, characterized in that using acid as degradation agent, by Sepia polysaccharide
It carries out degradation and obtains low molecular weight Sepia polysaccharide, the low molecular weight is the weight average molecular weight lower than 10kDa.
3. preparation method as claimed in claim 2, characterized in that the acid is trifluoroacetic acid.
4. preparation method as claimed in claim 2, characterized in that step: trifluoro is added into the aqueous solution of Sepia polysaccharide
Acetic acid, then heating reaction, obtains low molecular weight Sepia polysaccharide after reaction；
Preferably, reaction temperature is 60~100 DEG C；It is further preferred that the reaction time is 1~8h.
5. preparation method as claimed in claim 2, characterized in that sour concentration is 0.4~0.6mol/L；Preferably, it reacts
Temperature is 75~85 DEG C, 3.5~4h of reaction time.
6. preparation method as claimed in claim 2, characterized in that isolate and purify the material after reaction, the separation
The step of purifying are as follows: add water to filter by the dry materials after reaction, then by the material after drying, then by filtered liquid into
The separation of row gel filtration chromatography；
Preferably, specific steps are as follows:
(1) material after reaction is dried；
(2) water that 1 volume is added in the material after drying is dissolved, after filtering, is connected using 150 tomographic system of AKTA avant
Sephadex G-50 gel column is isolated and purified, and every pipe collects 2 volume eluents automatically；
(3) step (2) the 2nd~8 pipe eluent is collected to be freeze-dried；
It is further preferred that the 5th~8 pipe eluent of collection is freeze-dried in step (3).
7. a kind of low molecular weight Sepia polysaccharide that any preparation method of claim 2~6 obtains.
8. a kind of pharmaceutical composition, characterized in that including low molecular weight Sepia polysaccharide described in claim 1 or 7.
9. a kind of pharmaceutical preparation, characterized in that wanted including low molecular weight Sepia polysaccharide or right described in claim 1 or 7
Pharmaceutical composition described in asking 8 and pharmaceutically acceptable auxiliary material and/or carrier.
10. low molecular weight Sepia polysaccharide, pharmaceutical composition according to any one of claims 8 described in a kind of claim 1 or 7 or power
Benefit require 9 described in pharmaceutical preparation preparing anti-tumor drug and/or enhance immune drug in application.
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