CN1269525C - Agglutinin II protein of rhizome of king solomonseal, and application - Google Patents
Agglutinin II protein of rhizome of king solomonseal, and application Download PDFInfo
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Abstract
The present invention relates to a solomonseal rhizome agglutinin II protein which is prepared from stem tubers of solomonseal rhizome as raw materials. A preparation process orderly comprises the steps of the preparation of a crude product, the separation and the purification of affinity columns and the purification of molecular sieves. In the separation and the purification of affinity columns, mannose-Sepharose4B is used as an affinity absorbent, and mannose solution is used for desorption. The molecular sieves are SephacrylS-100 columns. After affinity chromatography samples dissolved by normal salt solution are dialyzed to equilibrium, the samples are applied to the columns. The samples on the columns are eluted by the normal salt solution and are partially collected. After the detection of ultraviolet absorption and detection activity at the position of 280 nm, active components are collected. The dialyzed and desalted active components are frozen and dried, and the purified solomonseal rhizome agglutinin II protein is obtained. With ACV as a positive control medicine, the present invention uses a CPE method and an MTT method to study the activity of the solomonseal rhizome agglutinin II protein for resisting herpes simplex viruses (HSV). The present invention proves that the solomonseal rhizome agglutinin II protein has strong inhibiting effect on human herpes simplex viruses, which is helpful to develop a new medicine.
Description
Technical field
The invention belongs to phytohemagglutinin and application thereof, particularly a kind of is the Rhizoma Polygonati agglutinin II albumen and the application thereof of feedstock production with this Chinese herbal medicine of liliaceous plant capsule silk Rhizoma Polygonati (Polygonatumcyrtonema Hua).
Background technology
Agglutinin (Lectin) be the non-enzyme of a class that extensively exists of occurring in nature, non-immunity source, have sugared binding specificity and can make sedimentary albumen of saccharide complex or glycoprotein.Be found to castor bean so far from first phytohemagglutinin, people have found and have separated hundreds of kind agglutinin.
Liliaceous plant capsule silk Rhizoma Polygonati (Polygonatum cyrtonema Hua.) is China's tradition Chinese herbal medicine, Bao Jinku etc. are in " journal of biological chemistry " (the 12nd the 2nd phase of volume, in April, 1996) paper of delivering on " purification of Rhizoma Polygonati agglutinin II and part property research " has been introduced a kind of Rhizoma Polygonati agglutinin II, this kind Rhizoma Polygonati agglutinin II is a kind of acidoglycoprotein, with plant Rhizoma Polygonati tuber is raw material, preparation method is: the Rhizoma Polygonati tuber is leached with normal saline solution, behind the ammonium sulfate precipitation, precipitation is dissolved in distilled water, the dialysis desalination, lyophilization obtains Rhizoma Polygonati agglutinin crude product, use pig thyroglobulin-Sepharose 4B affinity column separation and purification then, with the quick column purification of CM-Sepharose ion exchange, d peak with the CM post carries out the SephadexG-100 sieve chromatography, obtain four protein peaks, all there is strong agglutination activity at three peaks, back, difference called after Rhizoma Polygonati agglutinin I (PCL I), Rhizoma Polygonati agglutinin II (PCLII), Rhizoma Polygonati agglutinin III (PCLIII), wherein Rhizoma Polygonati agglutinin II content is more, detect through ultraviolet and blood coagulation activity, collect the active peak of II, the dialysis desalination, lyophilization gets Rhizoma Polygonati agglutinin II albumen.
Bao Jinku etc. are in " journal of biological chemistry " (the 12nd the 6th phase of volume, in December, 1996) paper of delivering on " stability and the biological activity research of Rhizoma Polygonati agglutinin II molecule " discloses Rhizoma Polygonati agglutinin II to lymphocytic mitogenic activity, producing PHA with Guangzhou is contrast, Rhizoma Polygonati agglutinin II to human peripheral lymphocyte's conversion ratio up to 97.6%, the cell division ratio reaches 18.3%, is a kind of extremely strong mitogenesis source.
Herpes simplex virus (Herpes simplex virus, HSV) be to cause modal virus in human virus's property disease, most of people carry this virus for a long time, the form that infects with incubation period is confined in some sensory ganglion, brought out by self or environmental factors and activate virus, cause the skin bleb infringement.Therefore, one of main contents of anti-HSV research becoming antiviral research.At present both at home and abroad the anti-HSV medicine of research is existing tens kinds, and only acycloguanosine (ACV) is comparatively effective but carry out the transition to the medicine that clinical treatment HSV infects, and has had been found that many persisters take place, and it is imperative therefore to seek new anti-HSV medicine.
Summary of the invention
The object of the present invention is to provide a kind of new method to prepare Rhizoma Polygonati agglutinin II albumen, and proof Rhizoma Polygonati agglutinin II albumen is inhibited to the human simple herpesvirus, so that develop the new medicine of a class.
Technical scheme of the present invention: adopt cytopathic-effect inhibition assay (CPE method, see Cotarelo M, CatalanP, Sanchez-Carrillo C, Menasalvas A, Cercenado E, Tenoriao A, Bouza E.Cytopathiceffect inhibition assay for determining the in-vitro susceptibility of herpessimplex virus to antiviral agents.J Antimicrob Chemother, 1999,44:705 ~ 708.) and mtt assay (see Campling BG, Pym J, Baker HM, Cole SP, Lam YM.Chemosensitivity testingof small cell lung cancer using the MTT assay.Br J Cancer, 1991,63 (1): 75-83.), with the positive control drug of acycloguanosine injection (ACV), research Rhizoma Polygonati agglutinin II albumen anti-herpes simplex virus (HSV) activity has proved that Rhizoma Polygonati agglutinin II albumen has very strong inhibitory action to the human simple herpesvirus.Therefore, Rhizoma Polygonati agglutinin II protein Preparation can be become the medicine of treatment or prevention herpes simplex virus associated diseases, administrated method can be by oral, intravenous injection etc.
Described Rhizoma Polygonati agglutinin II albumen is raw material with plant Rhizoma Polygonati tuber, and the adopting process steps in sequence is that the method for " crude product preparation, affinity column separation and purification, molecular sieve purification " obtains.The crude product preparation is the Rhizoma Polygonati tuber to be cut into lamellar leach with normal saline solution, mash with the normal saline solution leaching through high-speed tissue mashing machine, the gained supernatant adds ammonium sulfate precipitation, and precipitation is dissolved in distilled water again, the dialysis desalination, lyophilization promptly gets Rhizoma Polygonati agglutinin crude product; The affinity column separation and purification is that crude product is dissolved in normal saline solution, dialysis equilibrium, the centrifugal insoluble matter of removing, get supernatant and carry out affinity chromatograph, with mannose-Sepharose 4B is affinity adsorbent, with 0.1～0.3mol/L mannose solution desorption, fraction collection all in eluting and the desorption process, collecting pipe is after the 280nm place measures uv absorption, with the sample normal saline dialysis, with the agglutination activity of rabbit erythrocyte sample for reference, merge active part and dialyse and desalt, lyophilization gets the affinity chromatograph sample; Molecular sieve purification is an exchange column with Sephacryl S-100 post, with above-mentioned affinity chromatograph sample with physiological solt solution dissolving and dialysis equilibrium after upper prop, use the physiological solt solution eluting, fraction collection, after the 280nm place measures uv absorption and detection of active, collect active part, the dialysis desalination, lyophilization promptly obtains the Rhizoma Polygonati agglutinin II albumen of purification.
Beneficial effect of the present invention:
1, enlarged the proteic range of application of Rhizoma Polygonati agglutinin II, the viral disease that causes for the treatment herpes simplex virus provides a kind new medicine.
2, with respect to the method for providing of existing Rhizoma Polygonati agglutinin II, simplified technological process, shortened preparation time, thereby can reduce preparation cost.
3, during the affinity column separation and purification, mannose is higher than Elityran specificity of the prior art, and separating effect is better, and during molecular sieve purification, molecular sieve S-100 is quicker than G-100 of the prior art, is suitable for extensive, fast purifying.
Description of drawings
Fig. 1 is the proteic SDS-PAGE electrophoretogram of Rhizoma Polygonati agglutinin II of the present invention;
Fig. 2 is the active figure as a result of Rhizoma Polygonati agglutinin II albumen anti-herpes simplex virus II type of the present invention (HSV-II), among the figure, a: the cytopathy that normal cell, b:HSV-II cause, cde:10, the inhibitory action of 2.5,5 μ g/ml Rhizoma Polygonati agglutinin II (PCLII) pair cell pathological changes.
The specific embodiment
Embodiment 1: the proteic preparation of Rhizoma Polygonati agglutinin II
In the present embodiment, the proteic preparation method of Rhizoma Polygonati agglutinin II is as follows:
The preparation of 1 crude product
To be cut into flaky Rhizoma Polygonati tuber with normal saline solution leaching (G/V=1: 5) spend the night, mash through high-speed tissue mashing machine, the normal saline solution leaching is spent the night, filtered through gauze, centrifugal (4 ℃, 4500r/min, 30min), in supernatant, add solid ammonium sulfate and reach 30% saturation, put 4 ℃ and spend the night, centrifugal (4 ℃, 4500r/min, 30min) go precipitation, supernatant continues to add ammonium sulfate to 90%, centrifugal collecting precipitation, and precipitation is dissolved in distilled water, the dialysis desalination, lyophilization gets Rhizoma Polygonati agglutinin crude product.
2 affinity column separation and purification
Get crude product 200mg and be dissolved in the 20ml normal saline solution, dialysis equilibrium, the centrifugal insoluble matter of removing is got supernatant and is carried out affinity chromatograph (post: 2.6 * 40cm).With mannose-Sepharose 4B is affinity adsorbent, behind physiological solt solution balance chromatographic column, with above-mentioned agglutinin crude product upper prop, identical physiological solt solution be eluted to effluent in 280nm place absorption value less than 0.02, use 0.2mol/L mannose solution desorption instead, fraction collection all in eluting and the desorption process, flow velocity 18ml/hr, the 3-4ml/ pipe, collecting pipe is after the 280nm place measures uv absorption, with the sample normal saline dialysis, with the agglutination activity of rabbit erythrocyte sample for reference, merge active part and dialyse and desalt, lyophilization gets the affinity chromatograph sample.
3 molecular sieve purification
Learn from else's experience the Sephacryl S-100 dress post handled (1.6 * 100cm), use the normal saline balance.With above-mentioned affinity chromatograph sample with physiological solt solution dissolving and dialysis equilibrium after upper prop, use the same solution eluting, fraction collection, flow velocity are 2ml/min, 3ml/tube, after the 280nm place measures uv absorption and detection of active, collect active part, the dialysis desalination, lyophilization obtains the Rhizoma Polygonati agglutinin II of purification, detect to single protein component through SDS-PAGE, see Fig. 1.
Embodiment 2: Rhizoma Polygonati agglutinin II albumen is to the inhibitory action of human simple herpesvirus
1 material and method
1.1 material and instrument
Rhizoma Polygonati agglutinin II albumen: be prepared from by embodiment 1 described method.
Cell: African green monkey kidney cell (Vero cell line), preclinical medicine institute of West China medical courses in general portion of Sichuan University provides cell strain.Virus: herpes simplex virus I I type (HSV-II), 333 strains ,-70 ℃ of preservations provide strain by Wuhan Virology Institute,Chinan academy of Sciences.
Culture medium: RPMI-1640 (GIBCO product) is according to the explanation dissolving, be adjusted to pH7.2, the filtering with microporous membrane degerming, adding calf serum (Chengdu Harry's biological engineering company limited) to concentration is 10%, contains 100U/ml penicillin (Chongqing Yaoyou Pharmaceutical Co., Ltd.) and 100 μ g/ml streptomycin sulfates (Shanghai four medicine limited companies).
Trypsin: GIBCO product; MTT:3-(4,5)-two methyl-second-thiazole-(2,5)-dimethyl bromination tetrazole indigo plant, the Sigma product; 96 orifice plates: Nunclon product.
Positive control medicine: acycloguanosine injection (ACV), beneficial pharmaceutcal corporation, Ltd of beautiful pearl Hubei section of group.
Phosphate buffer PBS (does not contain Ca
2+, Mg
2+): be Na
2HPO
4And NaH
2PO
4The aqueous solution of the concentration 0.02M that is mixed with, pH=7.0, used chemical reagent are homemade analytical pure.
Microporous filter membrane: φ 25mm, the aperture is 0.22 μ m, Shanghai Institute of Pharmaceutical Industry; Microplate reader: Model 550, the Bio-Red product.
All cell culture articles for use use after 30 minutes through 121～126 ℃ of autoclave sterilizations.
Testing sample is dissolved with culture medium, and wherein not diffluent sample is then used a small amount of dimethyl sulfoxide (DMSO) hydrotropy, and the some concentration of culture medium doubling dilution are used in filtration sterilization then, places-20 ℃ of preservations.
1.2 method
1.2.1 the mensuration of Rhizoma Polygonati agglutinin II protein concentration
Measure with the Folin-phenol reagent, make standard with bovine serum albumin.
1.2.2 African green monkey kidney cell (Vero cell) is cultivated
The African green monkey kidney cell (Vero) that grows up to monolayer in the culture bottle is washed 3 times with PBS, added 0.25% trypsin solution, 1～2ml then and be digested to unicellularly, behind cell counting, be diluted to 1 * 10 with culture medium
5The cell suspension of cell/ml is seeded to 96 porocyte culture plates, every hole 0.2ml, 37 ℃, 5%CO
2Cultivated 24 hours in the incubator, treat to experimentize after the Vero cell grows up to monolayer.
1.2.3 the preparation of herpes simplex virus I I type (HSV-II) solution
Get frozen HSV-II solution one pipe, get 0.2ml after thawing and be inoculated in the 10ml culture bottle that grows up to cell monolayer, 37 ℃, 5%CO
2Absorption is 1 hour in the incubator, and the virus with the PBS flush away does not adsorb adds fresh culture 10ml, cultivates 2～3 days.Whole pathological changes of observation of cell but when not taking off wall as yet, inhale gently and go the upper strata culture medium, add fresh culture 10ml, blow down cell with dropper and become cell suspension, transfer in the 10ml centrifuge tube, place-70 ℃ of refrigerator multigelations three times, centrifugal 15 minutes of 4 ℃, 4000rps, packing supernatant 0.5ml/ pipe ,-70 ℃ of preservations.
Titration of virus (cytopathic effect method): get-70 ℃ of frozen viral liquid, room temperature is thawed, with 6 concentration of the continuous 10 times of dilutions of PBS, be inoculated on 96 orifice plates that grow up to cell monolayer, each concentration is done four multiple holes, adsorbs 1 hour, with PBS flush away viral adsorption not, add fresh culture, 37 ℃, 5%CO
2Incubator is cultivated observation of cell pathological changes effect after 72 hours.Calculate 50% TCID (median tissue culture infection dose, TCID
50).
1.2.4 the cytotoxicity experiment of Rhizoma Polygonati agglutinin II sample
The CPE method: treat that 96 orifice plate cells grow up to the monolayer hypsokinesis and go culture medium, by 4 multiple holes of each concentration, every hole adds 0.2ml through the Rhizoma Polygonati agglutinin II of serial doubling dilution solution, and control wells adds fresh culture 0.2ml, 37 ℃, 5%CO
2Incubator continues to cultivate 72h, inverted microscope following every day of observed and recorded result.
Mtt assay: behind the 72h, every hole adds 5mg/ml MTT solution 20 μ l, 37 ℃, 5%CO
2Incubator continues to cultivate 4 hours, as seen black-and-blue first a ceremonial jade-ladle, used in libation granule, inhale gently and remove supernatant, add the 0.2ml dimethyl sulfoxine, 5min vibrates on agitator, first a ceremonial jade-ladle, used in libation granule is fully dissolved, carry out dual wavelength (570 and 630nm) method with microplate reader and measure absorbance, calculate the half toxic concentration (CC of medicine pair cell according to Reed and Muench method
50).
1.2.5 the anti-HSV-II activity experiment of Rhizoma Polygonati agglutinin II sample
Treat that 96 orifice plate cells grow up to inhale behind the monolayer and go culture medium that get frozen viral liquid, after 100 times of PBS dilutions, the viral liquid 20 μ l that every hole adds dilution put 37 ℃, 5%CO
2Absorption is 1 hour in the incubator, the suction venom of preventing or cure a disease, by 3 multiple holes of each concentration, every hole 0.2ml adds through the Rhizoma Polygonati agglutinin II of serial doubling dilution sample solution, add the 0.5mg/ml ACV, the cell that add 0.2ml in fresh culture 0.2ml, the positive drug contrast simultaneously in the negative control and do not add viral liquid in contrasting, only add the 0.2ml fresh culture, 37 ℃, 5%CO
2Cultivated 72 hours in the incubator, the microscopy record is measured the O.D value with mtt assay simultaneously.Calculate the half-inhibition concentration (IC of medicine according to Reed and Muench method to virus
50) and therapeutic index (TI).
B=log (＜50% sample concentration) C=log (extension rate)
2 experimental results and discussion
2.1 virus virulence measurement result
The cytopathic effect method records tiring of HSV-II and is TCID
50=1 * 10
-4, with 100 times of PBS dilutions, be 100 * TCID during use according to virus quantity
50Inoculation, 20 microlitres are inoculated in every hole.
2.2 the anti-HSV-II activity research of Rhizoma Polygonati agglutinin II
2.2.1 with mtt assay research Rhizoma Polygonati agglutinin II cytotoxicity and anti-HSV-II activity
As positive control,, the results are shown in Table 1 and table 2 with 5ug/ml ACV with mtt assay research Rhizoma Polygonati agglutinin II cytotoxicity and anti-HSV-II activity.
The Vero cytotoxicity result of table 1 Rhizoma Polygonati agglutinin II
| Concentration (mg/ml) | 8 | 4 | 2 | 1 | 0.5 | 0.25 | 0.125 | 0.0625 | The cell contrast | CC 50 mg/ml |
| O.D meansigma methods ± s suppression ratio (%) | 0.453 0.02 33.8 | 1.145 0.03 84.8 | 1.214 0.01 89.9 | 1.245 0.04 92.2 | 1.220 0.03 90.4 | 1.291 0.03 95.6 | 1.301 0.02 96.4 | 1.312 0.04 97.2 | 1.350 0.02 | 5.29 |
The active result of the anti-HSV-II of table 2 Rhizoma Polygonati agglutinin II
| Concentration (ug/ml) | 4 | 2 | 1 | 0.5 | 0.25 | 0.125 | Virus control | The cell contrast | IC 50 ug/ml |
| O.D meansigma methods ± s suppression ratio (%) | 1.244 0.33 92.4 | 0.810 0.28 60.2 | 0.511 0.12 38.0 | 0.245 0.14 18.2 | 0.195 0.09 14.5 | 0.199 0.11 14.5 | 0.107 0.05 | 1.346 0.03 | 1.45 |
By table 1 can, Rhizoma Polygonati agglutinin II concentration is when 4mg/ml, cytopathy suppression ratio (being cell survival rate) has reached 84.8%, half cytotoxicity concentration is 5.29mg/ml.The O.D meansigma methods of 5ug/ml ACV positive control is 1.222 ± 0.05, and cell survival rate is 90.5%.From table 2 result as can be seen, Rhizoma Polygonati agglutinin II effect when concentration is 4ug/ml is suitable with 5ug/ml ACV.Its half toxic concentration (CC50) is 5.29mg/ml, and half-inhibition concentration (IC50) is 1.45ug/ml, and its therapeutic index TI is 3648.3, shows that Rhizoma Polygonati agglutinin II treatment safety is good.
2.2.2 cytopathic-effect inhibition assay (CPE) is measured the active result of the anti-HSV-II of Rhizoma Polygonati agglutinin II albumen
With culture medium Rhizoma Polygonati agglutinin II albumen is made into 1mg/ml concentration solution, filtration sterilization, and be diluted to respective concentration, carry out cytotoxicity and anti-HSV-II activity research.
● the proteic cytotoxicity of Rhizoma Polygonati agglutinin II
Through the observation of 72h, Rhizoma Polygonati agglutinin II albumen is 10
-1-10
-6All do not occur cytopathy under the dilution factor, show Rhizoma Polygonati agglutinin II no cytotoxicity under these concentration, the results are shown in Table 3.
Table 3 Rhizoma Polygonati agglutinin II is to the toxicity of Vero cell
| Hole number | PCLII (1000ug/ml) solution dilution multiple | Cell | |||||
| 10 -1 | 10 -2 | 10 -3 | 10 -4 | 10 -5 | 10 -6 | ||
| 1 2 3 | - - - | - - - | - - - | - - - | - - - | - - - | - - - |
Annotate: "-" represents acellular pathological changes; The cytopathy of "+" expression about 25%; The cytopathy of " ++ " expression about 50%; The cytopathy of " +++" expression about 75%; The cytopathy of " ++ ++ " expression 100%.(down together)
● the anti-HSV-II experimental result of Rhizoma Polygonati agglutinin II albumen
After cell inoculation virus and adding contain the proteic culture medium of Rhizoma Polygonati agglutinin II, observe, write down the cytopathy situation every day.After 72 hours, pathological changes does not appear in the cell blank matched group, and significant cytopathy appears in negative control group, and cytopathy does not appear in positive control medicine group, the results are shown in Table 4, table 5, table 6 and Fig. 2.
The anti-HSV-II experimental result of table 4ACV
| Hole number | AVC concentration (ug/mL) | |||||
| 1 2 3 | 500 - - - | 100 - - - | 50 - - - | 25 - - - | 12.5 - - - | 6.25 + + + |
The anti-HSV-II experimental result of table .5PCLII
| Hole number | PCLII concentration (ug/ml) | Cell | ||||||
| 1 2 3 | 10 - - - | 5 + + - | 2.5 + ++ + | 1.25 +++ ++ ++ | 0.625 +++ ++++ +++ | 0.3125 ++++ ++++ ++++ | HSV-II ++++ ++++ ++++ | - - - |
The active result of the anti-HSV-II of table 6 Rhizoma Polygonati agglutinin II
| Sample | The highest acellular malicious concentration (TC) | Minimum antiviral activity concentration (IC) | TC/IC |
| Rhizoma Polygonati agglutinin II (PCLII) | 1000ug/ml | 0.625ug/ml | 1600 |
As can be seen from Table 5, suppress the Vero cytopathy that HSV-II causes during greater than 10ug/mL fully when Rhizoma Polygonati agglutinin II concentration.Reduction with Rhizoma Polygonati agglutinin II concentration begins to take place cytopathy, and Rhizoma Polygonati agglutinin II sample concentration has 50% left and right sides cell that pathological changes has taken place when being 1.25ug/mL approximately, the anti-HSV-II activity of part is arranged, IC
50Between 1.25-2.50ug/ml.Its highest no cytotoxicity result and active result of minimum anti-HSV-II such as table 6.The highest acellular malicious concentration (TC) of sample is big more, and then the cytotoxicity of sample is more little; Minimum antiviral activity concentration (IC) is more little, and then antiviral effect is strong more.TC/IC is one of the potential antiviral safety of an assess sample roughly index, and TC/IC is big more, and then this secure sample is efficient.As can be seen from Table 6, the TC/IC of Rhizoma Polygonati agglutinin II sample is 1600, and the two differs 1600 times, shows that Rhizoma Polygonati agglutinin II is a kind of effectively external anti-HSV-II carbohydrate-binding protein.
Adopt mtt assay and CPE method to measure Rhizoma Polygonati agglutinin II and suppress the effect of HSV-II to the Vero cell infection, the conclusion unanimity that draws, IC
50Be respectively 1.45ug/ml and 1.25ug/ml, CC
50Be 5.29ug/ml, show that Rhizoma Polygonati agglutinin II can effectively resist HSV-II to Normocellular infection under low concentration, and Rhizoma Polygonati agglutinin II is very low to the toxicity of Vero cell, its therapeutic index is up to 3460, the TC/IC value also reaches 1600, illustrates that Rhizoma Polygonati agglutinin II is a kind of low toxicity biological activity carbohydrate-binding protein of external anti-HSV-II efficiently.Because Rhizoma Polygonati agglutinin II is a kind of carbohydrate-binding protein, can mutually combine with the sugar chain of HSV-cell surface, thereby the identification of sealing or blocking-up HSV-II and host cell and combine and reduce infection has the human II herpes simplex virus type clinical value of resisting.
Claims (1)
1, the application of Rhizoma Polygonati agglutinin II albumen in the medicine of preparation treatment or prevention herpes simplex virus associated diseases.
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| WO2023245415A1 (en) * | 2022-06-21 | 2023-12-28 | 四川大学 | Use of polygonatum cyrtonema hua. lectin in blocking invasion and infection of novel coronavirus |
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