CN108498533A

CN108498533A - A kind of medicinal usage of synanthrin

Info

Publication number: CN108498533A
Application number: CN201810714224.7A
Authority: CN
Inventors: 王顺春; 李宁; 施松善
Original assignee: Shanghai University of Traditional Chinese Medicine
Current assignee: Shanghai University of Traditional Chinese Medicine
Priority date: 2018-07-03
Filing date: 2018-07-03
Publication date: 2018-09-07

Abstract

The invention discloses a kind of medicinal usage of synanthrin, the medicinal usage refers to the drug for being used to prepare prevention using synanthrin as active constituent or sole active agent and/or treating Autoimmune Encephalomyelitis.Experiment shows that after giving EAE model mice synanthrin interventions its clinical score can be substantially reduced, and can delay the time of morbidity；It is significantly reduced it has also been found that giving inflammatory cell infiltration in the EAE model mice substantia alba medullae spinalis of synanthrin in terms of the histology, demyelinate phenomenon is also relieved；RT PCR results also show synanthrin and effectively reduce inflammation gene expression expression in EAE model mices myeloid tissue simultaneously；And with it to adjust Th17/Treg cell balances in Mice Body related for its therapy mechanism；Illustrate that synanthrin is expected to be used to prepare prevention and/or treats the drug of Autoimmune Encephalomyelitis, be especially expected to be used to prepare prevention and/or treat the drug of multiple sclerosis, there is extensive prospect in medicine.

Description

A kind of medicinal usage of synanthrin

Technical field

The present invention is to be related to a kind of medicinal usage of synanthrin, belongs to pharmaceutical technology field.

Background technology

Multiple sclerosis (Multiple sclerosis, MS) is one kind characterized by central nervous system inflammatory demyelinate Autoimmune disease, major clinical feature be sings and symptoms time and space it is multiple, First-episode more show as regarding Power decline, limb adynamia and sensory disturbance etc..MS influences about millions of people in the whole world, is cause young man to paralyse important Disease.It is now recognized that MS is to lead to the breaking-out of disease jointly by the factor of two aspects of h and E, and it is specific for its Pathogenesis, mainly think with CD4+T it is cell-mediated for the autoimmune response of central nervous system myelin protein it is related, Helper T lymphocyte subgroup especially Th17 cell subsets penetrates blood-brain barrier in CD4+T cells, penetrates into central nervous system, leads to Cross the inflammatory factors such as secretion IL-17, Il-22 and recruit other immunocytes such as macrophage etc., and then sustained activation brain is real The macrophage of microglia and colonization in matter, so as to cause inflammatory demyelinate and nerve retrograde affection；CD4+T cells Another subgroup Treg cells have the function of inhibiting excessive immune response, can be played to MS by secreting IL-10 and TGF-β Mitigation.The balance of Th17 cells and Treg cell proportions plays an important role in MS diseases.It is simulated in laboratory The animal model of multiple sclerosis is experimental allergic encephalomyelitis' model (Experiment Allergic Encephalomyelitis model, also known as：Experimental autoimmune encephalomyelitis model), abbreviation EAE models.

There are no very effective medicines for multiple sclerosis at present, clinically mainly using glucocorticoid and Interferon beta (IFN-β) and Glatiramer acetate etc. are treated (Li Mengqiu etc., the clinical evidence of Treatment of Multiple Sclerosis measure Evaluation, contemporary Chinese neurological disease magazine the 2nd phase of volume 12 in April, 2012).These drugs can only shorten more in most cases The hair property hardening course of disease, but progression of disease and healing disease can not be prevented, and these drugs have apparent secondary work mostly With since MS has the characteristics that chronic long, although the stronger medicine of toxic side effect has certain curative effect, long-term use also may be used Other harmful effects can be caused, therefore it is particularly significant to develop more safely and effectively MS medicines.

Synanthrin is that one kind passes through β -2, the levulan of 1 glucosides key connection, and conduct very wide in distributed in nature by fructose The depot polysaccharide of various plants, synanthrin have no adverse effects for human body, have been identified as a kind of functional food.Modern pharmacology Research finds that synanthrin has the function of that hypoglycemic can treat fat and diabetes, clinical trial and show to daily intake 16g chrysanthemums Sugar can significantly improve the blood glucose level and serum endotoxin level of obese women.

Up to now, there are no the relevant reports that synanthrin is used to prepare treatment Autoimmune Encephalomyelitis drug, more There is no synanthrin to be used to prepare the relevant report for the treatment of multiple sclerosis drug.

Invention content

In view of the above-mentioned problems existing in the prior art, the object of the present invention is to provide a kind of medicinal usages of synanthrin, to open up The medical value of wide synanthrin.

The medicinal usage of synanthrin of the present invention refers to using synanthrin as active constituent or sole active agent for making The standby drug prevented and/or treat Autoimmune Encephalomyelitis.

Furtherly, the medicinal usage of synanthrin of the present invention refers to using synanthrin as active constituent or sole active Ingredient is used to prepare prevention and/or treats the drug of multiple sclerosis.

In the drug, the molecular weight of synanthrin is 500~50000 dalton, preferably 2500~6000 dalton.

In the drug, the content of synanthrin is 0.1~99.9wt%, preferably 30~99wt%.

Effective administration dosage of active constituent of the present invention can be with drug used, the pattern of administration and disease to be treated Disease severity and change.

It obtains it will be understood by those skilled in the art that heretofore described synanthrin can be bought by commercial sources, can also lead to It crosses a variety of methods well known in the art, obtained using well known raw material, for example chemical synthesis or extracted from plant obtains.

The synanthrin of the present invention can be used alone or be used in the form of pharmaceutical composition.Pharmaceutical composition includes as work Property ingredient the present invention synanthrin and pharmaceutical acceptable carrier or other common excipient substances." pharmaceutical acceptable carrier " will not destroy this hair The pharmaceutical active of bright synanthrin, while its effective dose, dosage when can play pharmaceutical carrier effect are nontoxic to human body.

Described pharmaceutical acceptable carrier includes but not limited to：Soft phosphatide, aluminum stearate, aluminium oxide, ion exchange material, self-emulsifying Drug delivery system, tween or other surfaces activator, haemocyanin, buffer substance for example phosphate, amion acetic acid, sorbic acid, Water, salt, electrolyte such as sulfate protamine, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, magnesium silicate, saturated fatty acid Partial glyceride mixtures etc..Other common excipient substances include but not limited to：Adhesive (such as microcrystalline cellulose), filler (such as starch, glucose, Lactis Anhydrous and lactose bead), disintegrant (such as cross-linked pvp, crosslinked carboxymethyl fecula sodium, crosslinking carboxylic first Base sodium cellulosate, low-substituted hydroxypropyl cellulose), lubricant (such as magnesium stearate) and sorbefacient, absorption carrier, fragrance Agent, sweetener, excipient, diluent, wetting agent etc..

Drug of the present invention can give patient with various administration routes, including but not limited to oral, transdermal, muscle, Subcutaneous and intravenous injection.

The dosage form of drug of the present invention is unlimited, as long as active constituent can be made effectively to reach internal dosage form all Can be with, including：Tablet, film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, takes orally sugar coated tablet Liquid, mouth containing agent, granule, electuary, pill, powder, paste, sublimed preparation, suspension, pulvis, solution, injection, suppository, ointment Agent, emplastrum, creme, spray, drops, patch etc.；It is preferred that peroral dosage form, such as：Capsule, tablet, oral solution, granule, Pill, powder, sublimed preparation, paste etc..The peroral dosage form can contain common excipient, such as adhesive, filler, dilution Agent, tablet agent, lubricant, disintegrant, colorant, flavoring agent and wetting agent when necessary can be coated tablet.Suitable fills out It includes cellulose, mannitol, lactose and other similar fillers to fill agent；Suitable disintegrant includes starch, polyethylene pyrrole Pyrrolidone and starch derivatives, such as sodium starch glycollate；Suitable lubricant includes, such as magnesium stearate；Suitable medicine The acceptable wetting agent of object includes lauryl sodium sulfate.

Other than drug is made, the various foods such as antioxidant, pigment, enzyme preparation can also be added in the synanthrin of the present invention Health food is made by the conventional method of this field in product additive.

Compared with prior art, the present invention has following conspicuousness advantageous effect：

The result of study of the present invention is shown：It can obviously be dropped after the synanthrin intervention for giving EAE model mices 200mg/kg Its low clinical score, and the time of morbidity can be delayed；It has also been found that giving the EAE model mice ridges of synanthrin in terms of histology Inflammatory cell infiltration significantly reduces in marrow white matter, and demyelinate phenomenon is also relieved；RT-PCR results also show chrysanthemum simultaneously Sugar effectively reduce inflammation gene expression in EAE model mices myeloid tissue (including：Il-17a,Il-6,Tnf-α,Ccr-2,Ccr-6, Inos and Gm-csf) expression；And with it to adjust Th17/Treg cell balances in Mice Body related for its therapy mechanism；Illustrate synanthrin It is expected to be used to prepare prevention and/or treats the drug of Autoimmune Encephalomyelitis, be especially expected to be used to prepare prevention and/or controls The drug of multiple sclerosis is treated, there is extensive prospect in medicine.

Description of the drawings

Fig. 1 is the clinical score figure for treating EAE model mices in the embodiment of the present invention 1 with 200mg/kg synanthrin；Wherein, horizontal Coordinate is morbidity number of days (D), and ordinate is the clinical score (score) of disease；* represents P ＜ 0.01；

Fig. 2 is the incidence figure for treating EAE model mices in the embodiment of the present invention 1 with 200mg/kg synanthrin；Wherein, horizontal seat It is designated as morbidity number of days (D), ordinate is the incidence of disease；

Fig. 3 is to take each group mouse spinal cord after treating EAE model mice models in the embodiment of the present invention 1 with 200mg/kg synanthrin Effect after progress HE dyeing；Wherein, arrow meaning is exemplary inflammatory cell aggregation position；

Fig. 4 is to take each group mouse spinal cord after treating EAE model mice models in the embodiment of the present invention 1 with 200mg/kg synanthrin Effect after progress LFB dyeing；Wherein, arrow meaning is typical demyelination；

Fig. 5 is that RT-PCR detects each group after treating EAE model mice models in the embodiment of the present invention 1 with 200mg/kg synanthrin The result of mouse spinal cord tissue inflammation related gene expression；Wherein, Fig. 5 A to Fig. 5 G be successively inflammation gene expression Tnf- α, Il-6, The result of Il-17a, Inos, Ccr-2, Ccr-6 and Gm-csf expression；

Fig. 6 is to take its spleen lymphocyte after treating EAE model mices in the embodiment of the present invention 1 with 200mg/kg synanthrin, Carry out the streaming result figure after Th17 cells (CD4+IL-17+) dye；

Fig. 7 is to take its spleen lymphocyte after treating EAE model mices in the embodiment of the present invention 1 with 200mg/kg synanthrin, Carry out the streaming result figure after Treg cells (CD4+CD25+Foxp3+) dye；

Fig. 8 is the clinical score figure for treating EAE model mices in the embodiment of the present invention 2 with 200mg/kg synanthrin；Wherein, horizontal Coordinate is morbidity number of days (D), and ordinate is the clinical score (score) of disease.

Specific implementation mode

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number are calculated by weight.

Embodiment 1：Molecular weight is effect of the synanthrin of 5KDa in Autoimmune Encephalomyelitis

1. experimental animal

C57BL/6J mouse (female, 6-8 weeks) are purchased from Shanghai Slac Experimental Animal Co., Ltd..All zooperies are equal It obtains the animal ethics committee of Shanghai Univ. of Traditional Chinese Medicine and approves (license number：SZY201510002).

2.EAE modelings and animal packet

Mouse was raised by one week adaptability, started modeling：By prepared H37RA (tubercle bacillus) and MOG35-55 (myelin oligodendroglia glycoprotein segment) mixes, abundant vortex 10min or more, and it is milky suspension, emulsification to make solvent Solvent afterwards divides 3 points of hypodermic injections by every 0.1ml amount in mouse midspinal line second half section hind leg both sides and rear dorsal line, and In simultaneously and 48h pneumoretroperitoneums inject the pertussis toxin (PTX, 400ng/ only) of 0.1ml, blank control group mouse is not immune, Only injection 0.1ml H37RA, the immune same day are denoted as the 0th day.

Experimental animal is divided into two groups, every group 10, respectively model group (giving distilled water), synanthrin administration group (200mg/ Kg synanthrin), it is administered since the 0th day, successive administration 24 days.

3. clinical score standard

The same time carries out neurological deficit score to mouse since 1st day immune, using Knoz point systems, daily with for the moment Between give mouse scoring, be observed continuously scoring 24 days, specific standards of grading are as follows：

0 point normal asymptomatic；

1 point of tail portion loses tension, sagging parallel to the ground；

2 points of hind limb weakness, tail is completely weak and limp to mop floor；

3 points of hind legs are paralysed substantially, difficulty of creeping, and are reflected without reversion, and tail is completely weak and limp to mop floor；

4 points of hind limb paralysis and forelimb partial paralysis, difficulty of creeping, and reflected without reversion；

5 points of death；

Symptom between the two standard person in terms of scholar 0.5.

4 pathological sections

Under Animal Anesthesia state, mouse is fixed on tablet, mouse thoracic cavity is cut off, heart is made to be completely exposed, in the heart At point, 0.03ml heparin is injected, left ventricle injects PBS (phosphate buffered saline solution), cuts off right auricle of heart and releases blood, PBS perfusions It is internal to whole blood outflow, it changes 4%PFA (paraformaldehyde) into and is fixed, it is seen that four limbs, tail are swung, and body gradually becomes Firmly, continue perfusion 5min, keep body completely stiff, take out spinal cord front half section tissue, be put into 4%PFA solution and fix, overnight, Paraffin embedding, slice carry out H＆E dyeing and LFB dyeing.

5 lymphocytes prepare and streaming dyeing

Sterile taking-up mouse spleen, as on the culture dish for filling the cold PBS of 4ml, after slide grinding, by splenocyte suspension mistake Gained filtrate is centrifuged, is discarded supernatant by 70um cell screen clothes；600ul erythrocyte cracked liquids are added in cell precipitation, stand 2min and split After solving red blood cell, 4-5ml PBS are mended, cross 70um cell screen clothes, filtrate centrifugation, gained precipitation plus the piping and druming of RPMI1640 culture mediums are mixed It is even to get lymphocyte suspension；It is 4 × 106/ml that cell concentration is adjusted after cell count, is planted in 24 orifice plates, and cell is added Stimulant (cell stimulation cocktail are purchased from ebioscience companies of the U.S.) stimulation 5h, takes out cell and carries out Streaming dyes, and steps are as follows：Padding, cell are added dyeing liquor (PBS+2%FBS (fetal calf serum)) and are resuspended, centrifuge washing 2 Secondary, precipitation is added 50ul dyeing liquors and is resuspended, and CD4 the or/and CD25 antibody-solutions configured are added, and mild mixing, 4 DEG C are protected from light It is incubated 30min；Then intracellular dyeing is carried out, after cell is cleaned twice using dyeing liquor, 100ul fixers are added, room temperature is protected from light It is incubated 20min；The washing of 2ml rupture of membranes liquid is added, is repeated 2 times, centrifuges, discards supernatant, cell is resuspended using the rupture of membranes liquid of 100ul, Suitable IL-17, IFN-γ or Foxp3 fluorescence antibodies is added, is incubated at room temperature 20min；The washing of 2ml rupture of membranes liquid is added, adds 2ml dyeing liquors wash, and the cell after centrifugation is resuspended using 500ul dyeing liquors, machine in preparation.Flow cytometer is BD Products FACS verse。

6. statistical analysis

All experiments are at least in triplicate；Zoopery sample size is 10, and data value is indicated with average value ± standard error, is used Graphpad softwares are for statistical analysis, the significant difference between each group using one-way analysis of variance (ANOVA) and Student ' the t methods of inspection are analyzed, and p ＜ 0.05 are considered to have statistical significant difference.

7. experimental result

(1) EAE modelings and internal synanthrin intervene clinical score and incidence result is as depicted in figs. 1 and 2：

Fig. 1 is the clinical score figure for treating EAE model mices in the present embodiment with 200mg/kg synanthrin；Fig. 2 is this implementation The incidence figure for treating EAE model mices in example with 200mg/kg synanthrin, from Fig. 1 and Fig. 2：After model group mouse modeling in It falls ill from 7th day, and gradually aggravates successively, peak in the 18th day or so the state of an illness, and synanthrin administration group mouse invasion is serious Degree is significantly lower than model group, and incidence is also low compared with model group, illustrates to work as and gives the EAE chrysanthemums of model mice 200mg/kg concentration Sugar can be relieved the clinical score of EAE morbidities after intervening, and reduce the incidence of disease.

(2) H＆E coloration results are as shown in Figure 3：

Fig. 3 is to take each group mouse spinal cord to carry out after treating EAE model mice models in the present embodiment with 200mg/kg synanthrin Effect after HE dyeing, as seen from Figure 3：Occur apparent inflammatory cell infiltration in EAE model mices spinal cord, and administration group mouse goes out The region of existing inflammatory cell infiltration is few compared with model group, and inflammatory cell number is also considerably less than model group mouse, this shows synanthrin Administration effectively alleviates EAE model mice inflammation of the central nervous system.

(3) LFB coloration results are as shown in Figure 4：

Fig. 4 is to take each group mouse spinal cord to carry out after treating EAE model mice models in the present embodiment with 200mg/kg synanthrin Effect after LFB dyeing, as seen from Figure 4：Occurs apparent Demyelination in EAE model groups mouse spinal cord, synanthrin administration group is small Demyelinate phenomenon unobvious in mouse spinal cord illustrate that synanthrin effectively improves the pathological change of the inflammatory demyelinate of EAE mouse, this Phenomenon meets the disease severity on mice behavior.

(4) myeloid tissue's inflammation gene expression testing result is as shown in Figure 5：

Fig. 5 is that RT-PCR detects each group after treating EAE model mice models in the embodiment of the present invention 1 with 200mg/kg synanthrin Mouse spinal cord tissue inflammation related gene expression as a result, as seen from Figure 5：Inflammation base in the myeloid tissue of synanthrin administration group mouse Because the expression of Tnf- α, Il-6, Il-17a, Inos, Ccr-2, Ccr-6 and Gm-csf are obviously small less than EAE model groups Mouse prompts synanthrin administration to have significant protective effect for the inflammation of EAE central nervous system of mice.

(5) streaming result is as shown in Figure 6 and Figure 7：

Fig. 6 is to take its spleen lymphocyte after treating EAE model mices in the present embodiment with 200mg/kg synanthrin, is carried out Streaming result figure after Th17 cells (CD4+IL-17+) dyeing；Fig. 7 is to treat EAE moulds in the present embodiment with 200mg/kg synanthrin Its spleen lymphocyte is taken after type mouse, the streaming result figure after Treg cells (CD4+CD25+Foxp3+) dye is carried out, from figure 6 and he is 7 visible：Compared to the spleen lymphocyte of EAE model group mouse, the ratio of Th17 cells in administration group mouse lymphocyte Example significantly reduces, and the ratio of Th1 cells is without significant change；Increased with the Treg cell proportions of immune response effect are inhibited Height, the result shows synanthrin administrations to affect the proportional balancing method of Th17 cells and Treg cells in EAE mouse pathologic processes, says Bright synanthrin has potential therapeutic effect to multiple sclerosis.

8. conclusion

Complex chart 1 is to Fig. 7 as it can be seen that can be relieved EAE hairs after the synanthrin intervention for giving EAE 200mg/kg concentration The clinical score of disease, and reduce the incidence of disease；It has also been found that giving the EAE mouse spinal cord white matters of synanthrin in terms of histology Middle inflammatory cell infiltration significantly reduces, and demyelinate phenomenon is also obviously eased；And synanthrin is for the therapeutic effect of EAE and its The balance for affecting the immunocyte Th17 and Treg cells in EAE Mice Bodies is related, and therefore, synanthrin can be used for preventing or treat Autoimmune Encephalomyelitis and multiple sclerosis.

Embodiment 2：Molecular weight is effect of the synanthrin of 3.1KDa in Autoimmune Encephalomyelitis

Material and method

1. experimental animal

C57BL/6J mouse (female, 6-8 weeks) are purchased from Shanghai Slac Experimental Animal Co., Ltd..All zooperies are equal The animal ethics committee of Shanghai Univ. of Traditional Chinese Medicine is obtained to approve.

2.EAE modelings and animal packet

Mouse was raised by one week adaptability, started modeling：Prepared H37RA and MOG35-55 is mixed, fully Vortex 10min or more, it is milky suspension to make solvent, and the solvent after emulsification is by every 0.1ml amount after mouse midspinal line Point 3 points of hypodermic injections of half section of hind leg both sides and rear dorsal line, and in simultaneously and 48h pneumoretroperitoneums inject the pertussis toxin of 0.1ml (PTX, 400ng/ are only), blank control group mouse is not immune, only injects 0.1ml H37RA, and the immune same day is denoted as the 0th day.

Experimental animal is divided into two groups, every group 10, respectively model group (giving distilled water), 3.1KDa synanthrin administration groups (200mg/kg synanthrin) was administered since the 0th day, successive administration 24 days.

3. clinical score standard

0 point normal asymptomatic；

1 point of tail portion loses tension, sagging parallel to the ground；

5 points of death；

Symptom between the two standard person in terms of scholar 0.5.

4. statistical analysis

All experiments are at least in triplicate；Zoopery sample size is 8, and data value is indicated with average value ± standard error, is used Graphpad softwares are for statistical analysis, the significant difference between each group using one-way analysis of variance (ANOVA) and Student ' the t methods of inspection are analyzed, and p ＜ 0.05 are considered to have statistical significant difference.

5. experimental result

EAE modelings and 3.1KDa synanthrin intervene clinical score as shown in Figure 8：

Fig. 8 is the clinical score figure for treating EAE model mices in the present embodiment with 200mg/kg synanthrin, as seen from Figure 8：Mould It falls ill in the 7th day after type group mouse modeling, and gradually aggravates successively, peak in the 18th day or so the state of an illness, and 3.1KDa Synanthrin administration group mouse invasion severity is significantly lower than model group, and incidence is also low compared with model group, illustrates that molecular weight is The synanthrin of 3.1KDa also has the function of good prevention or treatment Autoimmune Encephalomyelitis and multiple sclerosis.

Finally need indicated herein be：The part preferred embodiment that the above is only the present invention, should not be understood as to this hair The limitation of bright protection domain, those skilled in the art's the above according to the present invention make some it is nonessential improvement and Adjustment all belongs to the scope of protection of the present invention.

Claims

1. a kind of medicinal usage of synanthrin, it is characterised in that：Refer to being used for using synanthrin as active constituent or sole active agent Prepare the drug for preventing and/or treating Autoimmune Encephalomyelitis.

2. the medicinal usage of synanthrin according to claim 1, it is characterised in that：Refer to using synanthrin as active constituent or only One active constituent is used to prepare prevention and/or treats the drug of multiple sclerosis.

3. the medicinal usage of synanthrin according to claim 1 or 2, it is characterised in that：In the drug, the molecule of synanthrin Amount is 500~50000 dalton.