CN108607092A - A kind of application of Radix Angelicae Sinensis albumen in preparing adjuvant therapy medicine - Google Patents
A kind of application of Radix Angelicae Sinensis albumen in preparing adjuvant therapy medicine Download PDFInfo
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Abstract
The present invention relates to natural traditional Chinese medicine albumen field, relate more specifically to a kind of application of Radix Angelicae Sinensis albumen in preparing adjuvant therapy medicine；The Radix Angelicae Sinensis albumen n end sequence is GIQKTEVEAPSTVSA.The Radix Angelicae Sinensis albumen is used to prepare adjuvant therapy medicine, and Radix Angelicae Sinensis albumen is given before chemotherapy can mitigate damage of the chemotherapeutic drugs Cisplatin to mouse spleen；Radix Angelicae Sinensis albumen is given before radiotherapy, can be made by peripheral white blood cell amount, thymus index, index and spleen index, the Spleen nodes quantity according to mouse（CFU‑S）135%, 103%, 23%, 219% and 180% is dramatically increased respectively with the CAT vigor of spleen; it can also be promoted by GST the and GSH Px vigor according to mouse spleen; improve splenic white pulp damage; its Global Macros effect is better than amifostine; it can make to be significantly reduced 94% by according to mouse weight loss rate, to improve its life quality.
Description
Technical field
The present invention relates to natural traditional Chinese medicine albumen field, relates more specifically to a kind of Radix Angelicae Sinensis albumen and preparing adjuvant therapy treatment
Application in drug.
Background technology
Chemotherapeutic drugs Cisplatin（DDP）It is one of the broad spectrum anticancer specific drug with clinical activity based on platinum, but because
Its selectivity is not high, has to tumour cell outside powerful lethal effect, also can inhibit the immune system using spleen as representative.
Radiotherapy is the effective means for killing tumour, carries the treatment of 2/3 tumour patient.But because radiotherapy is produced
Radiation injury side effect caused by raw free radical is big, has seriously undermined body part and whole resistance, patient is often
It is forced to interrupt radiotherapy because of the generation of these side effects.So the prevention of tumor radiotherapy side effect will directly affect radiotherapy energy
It is no lasting, it is one of the principal element for determining finally can thoroughly effect a radical cure tumour, is increasingly subject to the concern and attention of people.
Amifostine（AMFT）Also known as WR-2721, it is uniquely to go through applied to clinical radioprotector.Before radiotherapy
Use amifostine in advance, can kidney, marrow, heart, ear and nervous system caused by substantially reduced chemotherapeutics toxicity.
But during Clinical practice, amifostine is gradually found to have larger toxicity, and most of patient can be made dizzy, evil occur
The adverse reactions such as the heart, vomiting.Since amifostine is expensive, the side effect after use is many, in recent years its Clinical practice
It gradually decreases, or even exits Clinical practice.Therefore, new chemotherapy and radioprotector are developed in market in urgent need.
Radix Angelicae Sinensis [Angelica sinensis（Oliv.）Diels] be Umbelliferae angelica a kind of perennial herb plant
Object, the good medicine of blood circulation promoting and enriching, root can enter medicinal material, sweet Wen Zhirun.Radix Angelicae Sinensis is respectfully called as famous one of the Chinese herbal medicine in China
" king of medicine " all has the circulatory system, respiratory system, hemopoietic system, immune system and nervous system etc. certain effect.When
Summing up albumen has scavenging ability of DPPH free radical, and the proliferation of normal liver cell L-02 had both been remarkably promoted in pole（P<0.01）'s
Meanwhile and can pole significantly inhibit the proliferation of K562 Leukaemia（P<0.01）.
The present invention obtains Radix Angelicae Sinensis PR-10 family proteins from Chinese traditional medicine angelica --- ASPR.- 10 family of pathogenesis-related proteins
（PR-10）Albumen is distributed widely in each kind and each organ of plant, belongs to plant stress-resistance albumen, is coerced with plant resistant biology
Compel（Pathogen）With abiotic stress（Arid, heat, cold and salinity）Ability it is related.
The present invention provides Radix Angelicae Sinensis albumen and its applications in preparing adjuvant therapy medicine.
Invention content
The object of the present invention is to provide a kind of application of Radix Angelicae Sinensis albumen in preparing adjuvant therapy medicine, new to develop
Chemotherapy and radiotherapy protective agent.
To solve the above problems, the technical solution adopted by the present invention is as follows：
The preparation method of the Radix Angelicae Sinensis albumen is：
With 10 times of 0.05 mol/L Tris-HCl buffer solutions of volume（pH 8.0）Angelica sinensis is impregnated, 4 DEG C of extractings are overnight, secondary
Daily 4 layers of filtered through gauze residue, filtrate collect supernatant in 12000 rpm, 4 DEG C of centrifugation 10min.The supernatant of gained is carried out
80% ammonium sulfate precipitation collects precipitation, is dissolved in 0.05 mol/L Tris-HCl buffer solutions of 2 times of volumes（pH8.0）In, warp
Sephadex G-50 chromatograph post separation, collect second eluting peak, as destination protein ASPR, and molecular weight is 18.33 kDa,
It is GIQKTEVEAPSTVSA to obtain N-terminal sequence through sequencing.The Radix Angelicae Sinensis albumen can be used for preparing prevention radiation insult drug.
A concentration of 0.5-2 mg/mL of use of the Radix Angelicae Sinensis albumen；By the Radix Angelicae Sinensis ASPR albumen being sterile filtered with sterile
PBS or normal saline dilution to use concentration.Application method is continuous intramuscular injection or quiet before giving cis-platinum or radiotherapy
Arteries and veins injects 3 d of Radix Angelicae Sinensis ASPR albumen, every time 0.5 mL.
The advantage of the invention is that：
（1）The present invention is for the first time by Radix Angelicae Sinensis ASPR albumen for cis-platinum damage and acute radiation injury model.
（2）The invention demonstrates that Radix Angelicae Sinensis ASPR albumen has protective effect to splenic injury caused by cis-platinum, can mitigate suitable
Damage of the platinum to mouse spleen.
（3）The invention demonstrates that Radix Angelicae Sinensis ASPR albumen has protective effect for acute radiation injury, given before radiotherapy
Radix Angelicae Sinensis ASPR albumen can make by peripheral white blood cell amount, thymus index, index and spleen index, the Spleen nodes quantity according to mouse（CFU-
S）135%, 103%, 23%, 219% and 180% is dramatically increased respectively with the CAT vigor of spleen, can also be promoted by the GST according to mouse spleen
With GSH-Px vigor, improve splenic white pulp damage, Global Macros effect is better than amifostine, can make by aobvious according to mouse weight loss rate
Work reduces 94% to improve its life quality.
The particular use of drug of the present invention：
（1）It can be used for preventing cancer patient's side effect caused by chemotherapy or radiotherapy, such as induced lung injury, radioactivity hematopoiesis system
System damage and hepatic radiation-induced injury etc..
（2）It can be used for the daily maintenences radiation insult such as medical treatment and the operating personnel of Radioactive Sources Used In Industry, such as maintenance core work
When industry equipment and rescue radiation accident patient.
（3）It can be used for the cosmic ray injury that aerospace personnel prevents space.
（4）It can be used for preventing other oxidative damages.
The pharmacological action of drug of the present invention：
The present invention Radix Angelicae Sinensis ASPR albumen can dramatically increase by shine mouse spleen tissue CAT vigor, can also be promoted its spleen GST and
GSH-Px vigor improves the damage of its splenic white pulp, increases its Spleen nodes（CFU-S）And peripheral white blood cell amount, promote its spleen
And thymus index, its weight is maintained, to improve its life quality.
Description of the drawings
Fig. 1 Radix Angelicae Sinensis ASPR protein SDS-PAGEs.M：Albumen Marker；1：Radix Angelicae Sinensis albumen（ASPR）.
Fig. 2 Radix Angelicae Sinensis ASPR albumen damages cis-platinum the influence of mouse spleen.A, the apparent figure of mouse spleen；B, mouse spleen
Index n=5, compared with normal group,** P<0.01, compared with cis-platinum damages control group,# P<0.05。
Fig. 3 Radix Angelicae Sinensis ASPR albumen by according to mouse body weight growth rate to being influenced.N=10, compared with normal group,** P <
0.01, compared with radiocontrast group,## P <0.01。
Fig. 4 Radix Angelicae Sinensis ASPR albumen by according to mouse peripheral white blood cells to being influenced.N=10, compared with normal group,** P <
0.01, compared with radiocontrast group,# P<0.05,## P <0.01。
Fig. 5 Radix Angelicae Sinensis ASPR albumen by according to mouse thymus index to being influenced.N=10, compared with normal group,** P <0.01,
Compared with radiocontrast group,## P<0.01,# P <0.05。
Fig. 6 Radix Angelicae Sinensis ASPR albumen by according to mouse index and spleen index to being influenced.N=10, compared with normal group,** P <0.01,
Compared with radiocontrast group,# P <0.05。
Fig. 7 Radix Angelicae Sinensis ASPR albumen by according to apparent of mouse spleen and histologic characteristics to being influenced（× 200 times） n=10.
Fig. 8 Radix Angelicae Sinensis ASPR albumen by according to mouse Spleen nodes quantity to being influenced.N=10, compared with normal group,* P <
0.05, compared with radiocontrast group,## P <0.01。
Fig. 9 Radix Angelicae Sinensis ASPR albumen by according to mouse CAT activity levels to being influenced.N=10, compared with normal group,* P <
0.05, compared with radiocontrast group,# P <0.05。
Figure 10 Radix Angelicae Sinensis ASPR albumen by according to mouse GST activity levels to being influenced.N=10, compared with normal group,** P <
0.01。
Figure 11 Radix Angelicae Sinensis ASPR albumen by according to mouse GSH-Px activity levels to being influenced.N=10, compared with radiocontrast group,# P <0.05。
Specific implementation mode
The purifying of 1 Radix Angelicae Sinensis ASPR albumen of embodiment
With 10 times of 0.05 mol/L Tris-HCl buffer solutions of volume（pH 8.0）The Radix Angelicae Sinensis being cut into small pieces is impregnated, 4 DEG C stood
At night, about 15 h, secondary daily 4 layers of filtered through gauze residue, for filtrate in 12000 rpm, 4 DEG C of 10 min of centrifugation, the supernatant of gained is to work as
Return protein crude extract；Crude protein liquid carries out one step of ammonium sulfate precipitation（0-80%）, precipitates overnight simultaneously collects its protein precipitation, is dissolved in 2
0.05 mol/L Tris-HCl buffer solutions of times volume（pH 8.0）In, the lysate of ammonium sulfate precipitation is splined on through balance
Liquid（The Tris-HCl buffer solutions of 0.05 mol/L, pH 8.0）Sephadex G-50 chromatographic columns fully after balance, with balance
Liquid elutes and collects second eluting peak, through SDS-PAGE electrophoresis detection purity.
Experimental result：
Radix Angelicae Sinensis albumen is finally obtained through coarse extraction, one step of ammonium sulfate precipitation, SephadexG-50 gel permeation chromatographies in SDS-
Single band is shown as on PAGE, molecular weight is the Radix Angelicae Sinensis albumen of 18.33 KDa, as shown in Figure 1.The N-terminal sequence of the albumen is
GIQKTEVEAPSTVSA, 18.33 kDa of the molecular weight of albumen are PR-10 family proteins, are named as ASPR.
Embodiment 2：Radix Angelicae Sinensis ASPR albumen damages cis-platinum the protective action of mouse
1, experimental animal and grouping
Male mouse of kunming, is purchased from Wu Shi animals, and weight is 20 ± 2 g.Normal raising three days before experiment.Mouse is divided at random
At 5 groups, every group 5, each group and its processing are as follows：Normal group（CON）With cis-platinum damage group（DDP）The first life of 3 d of continuous injection
0.3 mL of brine is managed, after last gives 2 h, 0.3 mL of CON groups injecting normal saline, it is molten that DDP groups inject 0.5 mg/mL cis-platinums
0.3 mL of liquid（7.5 mg/kg）；Albumen dosing group is respectively ASPR1（0.5 mg/mL）+ DDP groups, ASPR2（1.25 mg/
mL）+ DDP groups and ASPR3（2.0 mg/mL）+ DDP groups, three groups of mouse inject 0.5 mL 3d of protein solution, are given in last respectively
After medicine 2h, then inject 0.3 mL of cisplatin solution.
2, Testing index --- index and spleen index
All mouse are put to death after 2 d of modeling, take out the spleen of mouse, after removing remained blood, carry out quality weighing and record,
And calculate its index.
Index and spleen index=（Weight before spleen weight/execution）×100%.
Experimental result：
As shown in Fig. 2, compared with normal group, the spleen of cis-platinum damage control group becomes smaller, compared with cis-platinum damages control group, each egg
White dosing group spleen becomes larger.The measurement result of each group mouse spleen index also indicates that, compared with normal group, cis-platinum damage control
The index and spleen index pole of group significantly reduces（P<0.01）, only normally organize 57.19%.Compared with cis-platinum damages control group, each albumen
Dosing group index and spleen index has rise, wherein Radix Angelicae Sinensis ASPR albumen middle dose group significantly to restore 40.35%（P<0.05）.
Therefore we select 1.25 mg/mL of optimum protein dose point to do next embodiment 3.
Embodiment 3：To the disposable radioprotective prevention effect of Systemic radiation mouse
1, experimental animal
Male mouse of kunming, is purchased from Wu Shi animals, and weight is 20 ± 2 g.Free water and feed, feed are special irradiation
Material, timing air draft.Experiment before raising three days it is without exception.
2, irradiation condition and experiment packet
Experiment is randomly divided into 5 groups with kunming mice：Normal group CON（Non-administration is not radiated）, radiocontrast group XRT（Only radiate
Non-administration）, amifostine group（AMFT+XRT）（200 mg/kg radiate preceding 30 min injections）, Radix Angelicae Sinensis ASPR protein groups（ASPR+
XRT）（1.25 mg/mL）, 3 d Radix Angelicae Sinensis ASPR albumen are continuously injected intraperitoneally in administration group before radiating, and it is laggard that last time injects 2 h
Row radiation.Radioactive source is X-ray, is 6 Gy according to dosage, and dosage rate is 2 Gy/min, carries out disposable full-body exposure.After irradiation
It is common to feed 7d.
3, Testing index
3.1 mouse weights increase
The measured body weight of all mouse is carried out before radiation, weigh mouse weight in the set time daily later and is recorded, and is calculated
Body weight increase situation in mouse 7 days.
Body weight growth rate=（Mouse weight before mouse weight-radiation in 7th day）Mouse weight × 100% before/radiation.
3.2 peripheral white blood cells change
All mouse carry out cervical dislocation execution after radiating 7 days.Mouse after execution carries out heart extracting blood, every mouse at once
20 μ l of blood are taken, with the leucocyte dilution of 380 ul（2.0 mL+1% gentian violets of glacial acetic acid, 1.0 mL, distilled water are settled to 100
ML, mixing）After dilution, dilution is taken to instill in tally, stand 2-3 min, it is aobvious to wait for that leucocyte sinking is placed on ordinary optical
White blood cell count(WBC) is carried out under micro mirror.
3.3 Thymus and Spleen index
After winning thymus gland and the spleen removal remained blood of mouse, weighing and the record of quality are carried out, and calculate its organ index.
Thymus index=（Weight before thymic factor D injection/execution）×100%.
Index and spleen index=（Weight before spleen weight/execution）×100%.
3.4 exogenous Spleen nodes methods measure mouse hematopoietic stem cell
With reference to classical exogenous Spleen nodes method.Part spleen is taken out, BouinShi liquid is placed in（Picric acid saturated aqueous solution 30
ML, 10% formaldehyde, 10 mL, 2 mL of glacial acetic acid）In fix 24 h, then make macroscopical Spleen nodes after being rinsed with 95% alcohol and count, that is, count
Number spleen convex surface tubercle.The ratio of BouinShi liquid and spleen volume summation is more than 5：1.
Apparent of 3.5 spleens and Histological assessment
It takes part spleen tissue to be fixed with 10% formalin solution, is then embedded in it in paraffin by organization embedding program；It is fixed
Afterwards, histotomy is cut into 4 μm of slices and is dyed with H ＆ E.
The detection of 3.6 spleen oxidation resistances
Part spleen is made to 10% tissue homogenate, 4000rpm centrifuges 20min, and supernatant is packed as prepare liquid, uses Nanjing respectively
Build up CAT in kit measurement homogenation of spleen tissue, GST and GSH-Px vigor.
4, statistical analysis：It is examined using the Student's t- in Excel 2013, withP<0.05 is significant difference,P<
0.01 is pole significant difference.
5, result
As shown in figure 3, compared with normal group, the 7th day after 6Gy Systemic radiations of mouse, weight is in negative growth state（P<
0.01）.Compared with radiocontrast group, before radiating pre-administration amifostine or Radix Angelicae Sinensis albumen all can pole significantly inhibit by according to mouse
Weight loss（P<0.01）, pre-administration amifostine group can make by according to mouse weight rate of descent reduce 59%（P<0.01）, give in advance
Medicine Radix Angelicae Sinensis albumin A SPR's is then reduced 94% by according to mouse weight rate of descent（P<0.01）.
As shown in figure 4, compared with normal group, radiocontrast group mouse peripheral blood leukocyte count pole is remarkably decreased（P<0.01）.With
Radiocontrast group is compared, and pre-administration amifostine can make to be dramatically increased 106% by according to murine interleukin number before radiating（P<
0.05）, pre-administration Radix Angelicae Sinensis albumin A SPR's is dramatically increased 135% by according to mouse blood leukocytes number（P<0.05）.As shown in figure 5, with
Normal group is compared, and radiocontrast group thymus index pole significantly reduces（P<0.01）.Compared with radiocontrast group, pre-administration A meter Fu
Spit of fland or Radix Angelicae Sinensis albumen can all make to be significantly improved by according to mouse thymus index（P<0.05,P<0.01）, but the effect of Radix Angelicae Sinensis albumin A SPR
Effect is better than amifostine.
As shown in fig. 6, compared with normal group, the mouse spleen index pole of radiocontrast group significantly reduces（P<0.01）.With
Radiocontrast group is compared, and pre-administration amifostine can make to be significantly improved 33% by the index and spleen index according to mouse（P<0.05）, before radiation
Pre-administration Radix Angelicae Sinensis albumen can make to be dramatically increased 23% respectively by according to mouse spleen index（P<0.05）.
The results are shown in Figure 7 for spleen histology pathological section, and the spleen red pulp white pulp of simple radiation group mouse is chaotic, without apparent
Boundary.Give amifostine or Radix Angelicae Sinensis albumen in advance and have different degrees of improvement result to this, it is seen that clear-cut it is complete
Whole white pulp.
As shown in fig. 7, the spleen surface of normal mouse is smooth, simple radiation group mice spleen surface irregularity occurs
The Spleen nodes vesicle of macroscopic protrusion, i.e. spleen colony forming units（CFU-S）.Give amifostine or Radix Angelicae Sinensis albumen in advance
Afterwards, all there are Spleen nodes in varying numbers by according to mouse spleen surface.After the processing of Bouin liquid, each group mouse spleen colony shape
It is as shown in Figure 8 at units.As shown in Figure 8, normally for group mouse without CFU-S, there is a small amount of CFU-S in radiocontrast group.With put
It penetrates control group to compare, gives amifostine in advance and the CFU-S amounts of mouse can be improved but do not have conspicuousness, pre-administration Radix Angelicae Sinensis albumen is then
Make to be increased twice or more by the CFU-S according to mouse.
By Fig. 9, Figure 10 and Figure 11 it is found that compared with normal group, CAT, GSH-Px and GST of simple radiation group mouse spleen
Vigor significantly reduces（P<0.05；P<0.01）.Compared with simple radiation group, amifostine or Radix Angelicae Sinensis ASPR albumen are given in advance
It is promoted to some extent by the antioxidase activity according to mouse spleen.Wherein give in advance amifostine to by according to mouse spleen CAT and
GSH-Px vigor is obviously improved effect, for also being had a certain upgrade effect by the GST vigor according to mouse spleen.Radix Angelicae Sinensis
ASPR albumen by the CAT vigor according to mouse spleen to being obviously improved effect, and effect is slightly better than amifostine, for by according to mouse
GSH-Px the and GST vigor of spleen also has a certain upgrade effect.
In conclusion giving Radix Angelicae Sinensis ASPR albumen in advance compared with radiocontrast group, before radiating can make by white according to mouse peripheral blood
Cell number dramatically increases 135%（P<0.05）, thymus index dramatically increases 103%（P<0.05）, index and spleen index dramatically increases 23%（P
<0.05）, CFU-S dramatically increases 219%（P<0.05）, improve significantly to splenic white pulp damage, the CAT of spleen can be made to live
Power dramatically increases 180 %（P<0.05）, finally make to be significantly reduced 94% by according to mouse weight loss rate（P<0.01）.
CFU-S is the important indicator for reflecting spleen hematopoiesis function, although Radix Angelicae Sinensis albumen is to spleen antioxidant system and spleen
The protecting effect of index is slightly weaker than amifostine, but it is more much better than than amifostine to the promotion effect of spleen CFU-S.In addition,
Radix Angelicae Sinensis albumen by the promotion effect according to the WBC of mouse, thymus index and CFU-S to being better than amifostine, finally to Systemic radiation
The inhibiting effect of caused weight loss is also better than amifostine.Mouse weight growth rate can intuitively reflect the life of mouse
Quality is deposited, is the overall target of mouse radiation insult degree.Therefore, integrated protection effect of the Radix Angelicae Sinensis ASPR albumen to radiation insult
It is better than amifostine.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification should all belong to the covering scope of the present invention.
SEQUENCE LISTING
<110>University of Fuzhou
<120>A kind of application of Radix Angelicae Sinensis albumen in preparing adjuvant therapy medicine
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 15
<212> PRT
<213>Radix Angelicae Sinensis albumen
<400> 1
Gly Ile Gln Lys Thr Glu Val Glu Ala Pro Ser Thr Val Ser Ala
1 5 10 15
Claims (3)
1. a kind of application of Radix Angelicae Sinensis albumen in preparing adjuvant therapy medicine.
2. application according to claim 1, it is characterised in that：The Radix Angelicae Sinensis albumen n end sequence is
GIQKTEVEAPSTVSA, 18.33 KDa of molecular weight.
3. application according to claim 1, it is characterised in that：The Radix Angelicae Sinensis albumen is used to prepare Prophylactic chemotherapy, radiation insult
Drug.
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