CN102643316B - A kind of acylated glycosides compound and its preparation method and application - Google Patents
A kind of acylated glycosides compound and its preparation method and application Download PDFInfo
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- CN102643316B CN102643316B CN201110404825.6A CN201110404825A CN102643316B CN 102643316 B CN102643316 B CN 102643316B CN 201110404825 A CN201110404825 A CN 201110404825A CN 102643316 B CN102643316 B CN 102643316B
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- paeoniflorin
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Abstract
The present invention provides a class acylated glycosides compound and its preparation method and application, it is characterized in that it has following general structure：R1=H, OH or OAc, R2=p-toluenesulfonyls, salicyl.Acylated glycosides compound of the invention has immunoregulation effect, can be used to preparing rheumatoid arthritis medicine, and the various immunity diseases such as autoimmune disease and allergy such as preparation system lupus erythematosus, eczema, psoriasis medicine.
Description
Technical field
The present invention relates to a class acylated glycosides compound, and such acylated glycosides compound preparation method and application.
Background technology
The autoimmune diseases such as rheumatoid arthritis, systemic loupus erythematosus, eczema, psoriasis and allergy etc. are each
Immunity disease is planted, there is great harmfulness to the mankind.TGP is that a kind of to treat Rheumatoid Arthritis definite
Medicine, it may also be used for systemic lupus erythematosus, eczema, psoriasis, ankylosing spondylitis etc..Pharmacology and clinical research are sent out
Existing, TGP has a multipath suppression of autoimmune responses, and anti-inflammatory, analgesic, liver protection and to suppress autoimmunity etc. various
Pharmacological action.The main pharmacodynamics composition of TGP is one group of glycoside material, including Paeoniflorin, Hydroxy peoniflorin, peony
Glycosides, albiflorin, benzoylpaeoniflorin etc., wherein main component are Paeoniflorin, and its content is more than 40%.To Paeoniflorin
Pharmacokinetic shows that the gastrointestinal absorption of Paeoniflorin is very poor, and its bioavilability is very low, and eliminates fast.
Paeoniflorin title is as follows with structural formula：
English name：Paeoniflorin
Chemical name：5beta- [(Benzoyloxy) methyl] tetrahydro-5-hydroxy-2-methyl-2,5-
Methano-1H-3,4-dioxacyclobuta [cd] pentalen-1alpha (2H)-yl-beta-D-
glucopyranoside
Molecular formula：C23H28O11
Molecular weight：480.45
Structural formula is as follows：
The content of the invention
The purpose of the present invention provides one kind and can be used to prepare rheumatoid arthritis medicine, and system aiming at above-mentioned situation
The medicine of the various immunity diseases such as autoimmune disease and allergy such as standby systemic loupus erythematosus, eczema, psoriasis
A class acylated glycosides compound, it is characterized in that it has following general structure：
Formula one：
R1=H, OH or OAc；R2=p-toluenesulfonyls, salicyl
Formula two：
R1=H, OH or OAc；R2=p-toluenesulfonyls, salicyl
A kind of preparation method of above-mentioned acylated glycosides compound, it is made up of the following steps：
Step one,
R1=H, OH or OAc
The dissolving of said structure glycoside in organic solvent, the acylate groups such as p-toluenesulfonyl or salicyl are added
Organic solution, after stirring, fully reaction, appropriate water washing, organic solvent extraction, by organic solvent extract anhydrous sodium sulfate
Dry, filtering is evaporated.Absolute ethyl alcohol is recrystallized, and obtains pulverulent solids.
Step 2,
The pulverulent solids that step one is obtained are dissolved with a small amount of organic solvent, silica gel column chromatography is eluting, obtains powdered
Solid, acylated glycosides compound as of the invention.
Polyarthirtis index, secondary lateral joint of the acylated glycosides compound of the invention to rat assist agent arthritis
Swelling, the body weight for declining, elevated index and spleen index, elevated macrophages secrete IL-1 levels, enhanced T, bone-marrow-derived lymphocyte increase
Growing the aspects such as reaction and joint pathology morphologic change has different degrees of improvement result.Show acylated glycosides chemical combination of the invention
Thing has immunoregulation effect, can be used to prepare rheumatoid arthritis medicine, and preparation system lupus erythematosus, eczema, silver
The medicine of the various immunity diseases such as autoimmune disease and allergy such as bits disease.
Brief description of the drawings
Fig. 1, Paeoniflorin uv-spectrogram
Fig. 2, Paeoniflorin infared spectrum
Fig. 3, Paeoniflorin tolysulfonyl compound uv-spectrogram
Fig. 4, Paeoniflorin tolysulfonyl compound infared spectrum
Fig. 5, Paeoniflorin tolysulfonyl compound nuclear magnetic resonance map
Fig. 6, blank plasma HPLC collection of illustrative plates
Fig. 7, Paeoniflorin aqueous solution HPLC collection of illustrative plates
Fig. 8, Paeoniflorin tolysulfonyl compound methanol solution HPLC collection of illustrative plates
Fig. 9, Paeoniflorin+Paeoniflorin tolysulfonyl compound plasma sample HPLC collection of illustrative plates
Specific embodiment
The synthesis of embodiment 1, Paeoniflorin tolysulfonyl compound
Accurately weigh Paeoniflorin 2g to be dissolved in pyridine (10ml), be slowly added dropwise containing paratoluensulfonyl chloride in ice-water bath
The dichloromethane solution (5ml) of (3.5g, 18mmol), after stirring reaction 10h, is first washed twice, then use 200ml second with 500ml
Acetoacetic ester is extracted, and takes the appropriate anhydrous sodium sulfate drying of organic layer.A period of time is stood, filtering, revolving.Finally use absolute ethyl alcohol
Light yellow powdery solid crude product 0.51g is recrystallized to obtain, yield is 25.5%.
The purifying of embodiment 2, Paeoniflorin tolysulfonyl compound
Paeoniflorin tolysulfonyl compound crude product 0.5g accurately is weighed, is dissolved with a small amount of eluent, be slowly loaded with dropper
In (column chromatography uses the silica gel 120g, silicagel column to be on silicagel columnGlass column, wet method dress post).With dichloromethane-
Methyl alcohol (15: 1, V/V) is eluted, depending on eluent consumption is according to separating effect, while coutroi velocity, generally 5.0mlmin-1。
The TLC legal times track identification, obtain Paeoniflorin tolysulfonyl compound 0.32g, and yield is 64.0%.
The synthesis of embodiment 3, albiflorin tolysulfonyl compound
Accurately weigh albiflorin 0.5g to be dissolved in pyridine (8ml), be slowly added dropwise in ice-water bath containing to toluene sulphur
The dichloromethane solution (5ml) of acyl chlorides (1g, 9mmol), after stirring reaction 10h, is first washed twice, then use 100ml second with 100ml
Acetoacetic ester is extracted, and takes the appropriate anhydrous sodium sulfate drying of organic layer.A period of time is stood, filtering, revolving.Finally use absolute ethyl alcohol
Recrystallize to obtain light yellow powder solid crude product.
The purifying of embodiment 4, albiflorin tolysulfonyl compound
Albiflorin tolysulfonyl compound crude product accurately is weighed, is dissolved with a small amount of eluent, be slowly loaded with dropper
In (column chromatography uses the silica gel 120g, silicagel column to be on silicagel columnGlass column, wet method dress post).With dichloromethane-
Methyl alcohol (15: 1, V/V) is eluted, depending on eluent consumption is according to separating effect, while coutroi velocity, generally 5.0mlmin-1。
The TLC legal times track identification, obtain albiflorin tolysulfonyl compound.
The synthesis of embodiment 5, Paeoniflorin bigcatkin willow acylate
Accurately weigh Paeoniflorin 2g to be dissolved in pyridine (10ml), the dichloro containing bigcatkin willow acyl chlorides is slowly added dropwise in ice-water bath
Dichloromethane (5ml), after stirring reaction 10h, is first washed twice with 400ml, then is extracted with 150ml ethyl acetate, takes organic layer
Use appropriate anhydrous sodium sulfate drying.A period of time is stood, filtering, revolving.Finally buff powder is recrystallized to obtain with absolute ethyl alcohol
Shape solid crude product.
The purifying of embodiment 6, Paeoniflorin bigcatkin willow acylate
Paeoniflorin bigcatkin willow acylate crude product accurately is weighed, is dissolved with a small amount of eluent, silicagel column is slowly loaded onto with dropper
It is upper that (column chromatography uses the silica gel 120g, silicagel column to beGlass column, wet method dress post).With methylene chloride-methanol (15:
1, V/V) elute, depending on eluent consumption is according to separating effect, while coutroi velocity, generally 5.0mlmin-1.TLC is legal
When tracking identification, obtain Paeoniflorin bigcatkin willow acylate.
The synthesis of embodiment 7, albiflorin bigcatkin willow acylate
Accurately weigh albiflorin 0.5g to be dissolved in pyridine (8ml), acyl chlorides containing bigcatkin willow is slowly added dropwise in ice-water bath
Dichloromethane solution (5ml), after stirring reaction 10h, first with 100ml wash twice, then with 100ml ethyl acetate extraction, take
The appropriate anhydrous sodium sulfate drying of organic layer.A period of time is stood, filtering, revolving.Finally recrystallize pale yellow with absolute ethyl alcohol
The powdered solid crude product of color.
The purifying of embodiment 8, albiflorin tolysulfonyl compound
Albiflorin bigcatkin willow acylate crude product accurately is weighed, is dissolved with a small amount of eluent, silicon is slowly loaded onto with dropper
(column chromatography uses the silica gel 120g, silicagel column to be on glue postGlass column, wet method dress post).Use methylene chloride-methanol
(15: 1, V/V) is eluted, depending on eluent consumption is according to separating effect, while coutroi velocity, generally 5.0mlmin-1。TLC
Legal time tracking identification, obtains albiflorin bigcatkin willow acylate.
The Structural Identification of Paeoniflorin tolysulfonyl compound
Tolysulfonyl compound carries out ultraviolet, infrared, mass spectrum and nuclear magnetic resoance spectrum map analysis.Structural formula is as follows：
Paeoniflorin tolysulfonyl compound structural formula
Nuclear magnetic resonance data parsing (the 500MHz in CDCl of Paeoniflorin tolysulfonyl compound3)
The assay method of the bioavilability of Paeoniflorin and Paeoniflorin acylate
1st, the treatment of plasma sample：
The μ l of plasma sample 60 are taken, is added in 0.5ml centrifuge tubes, 180 μ l methyl alcohol of addition, vortex oscillation 3min protein precipitations,
12000r·min-1Centrifugation 10min, takes the μ l of supernatant 150 in another 0.5ml centrifuge tubes, adding the acetic acid of equivalent 0.02% water-soluble
Liquid, is vortexed and mixes.
2nd, while determining the analysis method of Paeoniflorin and Paeoniflorin tolysulfonyl compound in blood plasma：
Paeoniflorin is prepared to Methyl benzenesulfonyl compound PEG400 standard liquids：Precision weighs 100.79mg Paeoniflorins to methyl
Benzene sulfonyl compound is settled to scale in 10ml volumetric flasks with PEG400 solution, mixes, and stands, and obtains 10.079mg/ml Paeoniflorins
To Methyl benzenesulfonyl compound PEG400 standard liquids.
Paeoniflorin standard aqueous solution is prepared：Precision weighs 30.3mg Paeoniflorins in 10ml volumetric flasks, uses ultra-pure water constant volume
To scale, mix, stand, obtain 3.03mg/ml Paeoniflorin standard aqueous solutions.
3rd, chromatographic condition：
HPLC：Agilent 1200series；Agilent Zorbax SB-C18 posts (4.6 × 250mm, 5 μm)；C18In advance
Post；Mobile phase：Methyl alcohol -0.02%HAC aqueous solution gradient elutions；Column temperature：30℃；Detection wavelength：230nm；Flow velocity：1.0ml/
Min, sample size：66μl.
Gradient elution：
| Time (min) | Methyl alcohol/% | The 0.02%HAC aqueous solution/% |
| 0 | 35 | 65 |
| 8 | 35 | 65 |
| 32 | 57 | 43 |
| 36 | 57 | 43 |
4th, standard curve：
With Paeoniflorin PC (μ g/ml) as ordinate, peak area is abscissa, carries out linear regression, and gained is returned
Curve is：C=0.0095A+0.0226, as a result shows, in the range of 0.0498 μ g/ml~6.375 μ g/ml, blood plasma Paeoniflorin
Linear relationship is good between concentration and peak area, correlation coefficient r=0.9984.
With Paeoniflorin tolysulfonyl compound PC (μ g/ml) as ordinate, peak area is abscissa, is carried out linear
Return, gained regression curve is：C=0.0094A+0.0379, as a result shows, in 0.0154 μ g/ml~7.874 μ g/ml scopes
Interior, linear relationship is good between blood plasma Paeoniflorin concentration and peak area, correlation coefficient r=0.9982.
Relative bioavailability
280~350g SD rats, are administered orally by every group 5.Paeoniflorin distillation water dissolves, dosage 500mg/kg
Body weight；Paeoniflorin tolysulfonyl compound PEG400 dissolves, dosage 660mg/kg body weight.It is dense that HPLC methods determine blood medicine
Degree, measurement result calculates AUC with DAS3.1 softwares(0-∞), it is as a result as follows：
The AUC of Paeoniflorin(0-∞)：1085±625.2h·ng·ml-1
The AUC of Paeoniflorin tolysulfonyl compound(0-∞)：7817±1851h·ng·ml-1, relative bioavailability=
5.46
The AUC of Paeoniflorin in Paeoniflorin tolysulfonyl compound(0-∞)：8383h·ng·ml-1, relative bioavailability=
5.85
The test of pesticide effectiveness of the Paeoniflorin tolysulfonyl compound to rat assist agent arthritis
1st, purpose：Drug effect of the Paeoniflorin tolysulfonyl compound to rat assist agent arthritis.
2nd, animal：SD rats：Body weight 180+20g, male
3. method
3.1 experiment packets：SD rats, be randomly divided into 4 groups, i.e. normal group, model group, Paeoniflorin tolysulfonyl compound and
The strong group of positive control drug.Every group 12.
3.2AA models are induced：Rat foot claw intracutaneous injection freund adjuvant (CFA) 0.1ml, while setting saline control
Group.Medication, medication 10 days after inflammation appearance.The compound method of CFA：By in 10mg BCG vaccines addition 1ml paraffin oils, fully mix
Rearmounted 4 DEG C of refrigerator overnights, are fully mixed again before injection.
3.3 administration times and administrated method：After induction, sufficient pawl swelling is observed daily, redness occurs in sufficient pawl scoring, sufficient pawl,
Start medication.Gastric infusion, once a day, medication 12 days.D28, puts to death rat, observes pharmacodynamics index.
3.4 Testing index：
(1) measure of sufficient pawl swelling degree：With sufficient pawl capacity measurer in cause inflammation before, cause inflammation after d12, d16, d20, d24,
D28 periodic detection rat foot claw swellings.
(2) whole body pathology：Changes of weight, the incidence and severity of forelimb, ear and afterbody lesion.
(3) articular index：Scored by 0~4 Pyatyi, 0=is without redness；The small toe joints of 1=slightly swell；2=toe joints and vola pedis
Swelling；Sufficient pawl swelling below 3=ankle-joints；4=includes the whole sufficient pawl swellings including ankle-joint.
(4) pain index：Every mechanical threshold of pain of rat is determined with tenderness instrument.First stimulated using certain dynamics, if not having
Contracting leg reacts, then select a dynamics thereon to stimulate hallux, if there is contracting leg to react, selects its next dynamics to stimulate hallux,
Occur once with a preceding differential responses (have contracting leg react to without contracting leg react or without contracting leg react to have contracting reaction) when, i.e.,
The dynamics be can record as the mechanical threshold of pain (being designated as "None" tenderness more than 25g).
(5) thymus index, spleen index；Claim rat body weight, take off neck and put to death rat, peel off thymus gland and spleen, weigh respectively, with
The weight of thymus gland or spleen and obtains thymus index or index and spleen index than the body weight of upper rat, as a result with thymus index or spleen
Exponential representation.
(6) T of LPS and ConA inductions and B lymphocyte proliferation reaction, it is aseptic to take spleen RPMI1640 to prepare lymph thin
Born of the same parents' suspension, on 96 well culture plates, 100 μ l cell suspensions (final concentration of 5 × 10 is added per hole6·ml-1) and LPS (final concentrations
4mg·L-1) or ConA (final concentration 3mgL-1), whole volume is 200 μ l, respectively sets 3 multiple holes, puts 37 DEG C, 5%CO2Incubator is trained
Support 48h, induction T, B lymphocyte proliferation.Terminate 2h before culture, add 20 μ l MTT, culture to collect cell after terminating per hole, from
Supernatant is abandoned in heart 500g × 10min, suction, and 120ml glacial acetic acid is added per hole, is shaken 30 seconds, and selection wavelength is examined for the ELIASA of 570nm
Its absorbance (A) is surveyed, is as a result represented with the average of 3 multiple holes A.
(7) IL-1 is induced and detection
Prepare rat peritoneal macrophages suspension (2 × 106·ml-1), add 24 well culture plates, per hole 1ml, put 37 DEG C,
5%CO2Incubator culture 2h, abandons or adopts supernatant, is washed 3 times with D-Hank`s liquid, removes non-adherent cell, obtains cell monolayer, so
It is each afterwards to add 500 μ l RPMI1640 and LPS (final concentration 4mgL-1), put 37 DEG C, 5%CO2Incubator culture 48h, induction is produced
Raw IL-1, collects supernatant (containing IL-1 activity), and -20 DEG C of preservations are to be measured.
It is aseptic to take C57BL/6J mouse thymus, thymus cell suspension (5 × 10 is prepared with RPMI16406·ml-1), in 96 holes
On culture plate, 100 μ l cell suspensions, 50 μ l ConA (final concentration 3mgL are added per hole-1) dilution factors different with 50 μ l it is to be measured
Supernatant, respectively sets 3 multiple holes, puts 37 DEG C, 5%CO2Incubator culture 48h, terminates 2h before culture, and 20 μ l MTT, training are added per hole
Support and cell is collected after terminating, 500g × 10min is centrifuged, supernatant is abandoned in suction, and 120ml glacial acetic acid is added per hole, is shaken 30 seconds, selects ripple
The ELIASA of a length of 570nm detects its absorbance (A), is as a result represented with the average of 3 multiple holes A.
(8) IL-2 is induced and measure
The aseptic thymus gland RPMI1640 that takes prepares T lymphocyte suspensions, on 24 well culture plates, adds 500 μ l thin per hole
Born of the same parents' suspension (final concentration of 5 × 106·ml-1) and 500 μ l ConA (final concentration 3mgL-1), whole volume be 1ml, put 37 DEG C, 5%
CO2After incubator culture 48h, induced rat T lymphocyte produce IL-2, culture to terminate, it is centrifuged (2000rpm, 10min), receives
Supernatant (containing IL-2 activity) is taken, -20 DEG C of preservations are to be measured.
It is aseptic to take C57BL/6J mouse spleens, prepare splenocyte suspension (2 × 10 with RPMI16406·ml-1), take 10ml and put
Enter in blake bottle, add ConA (final concentration 3mgL-1), put 37 DEG C, 5%CO2Incubator culture 48h, centrifugation (1500rpm,
10min), take cell to be separately added into the layering liquid for filling 4ml, be centrifuged (1500rpm, 10min), then draw cell monolayer use
PBS liquid is washed 2 times, then is washed 1 time with RPMI 1640, is made into 1 × 107·ml-1Cell suspension, by the μ l of every hole 100,50 μ l
ConA (final concentration 3mgL-1) dilution factors different with 50 μ l supernatant to be measured, add 96 well culture plates in.3 multiple holes are respectively set, is put
37 DEG C, 5%CO2Incubator culture 24h, terminates 2h before culture, adds 20 μ l MTT, culture to collect cell culture after terminating per hole
After end, 500g × 10min is centrifuged, supernatant is abandoned in suction, and 120ml glacial acetic acid is added per hole, is shaken 30 seconds, selection wavelength is 570nm
ELIASA detect its absorbance (A), as a result represented with the average of 3 multiple holes A.
(9) joint pathology inspection.
Rat is put to death, taking metapedes inflammatory ankle-joint carries out HE dyeing, observes the pathological change of inflamed joints tissue：According to tight
Weight degree scores synovial hyperplasia, inflammation, pannus, cartilage destruction.
Inflammatory score：Scored by 0~3 level Four, 0=is normal；The less cell infiltration of 1=soft tissues；2=moderate inflammatory cells
Infiltration；The obvious cell infiltrations of 3=.
Cartilage destruction scores：By 0~5 six grades of scorings, 0=is normal；1=Toluidine blue stainings, slight cartilage is lost, without bright
The former division of present gum and cartilage cell lose.2=Toluidine blue stainings, slight cartilage is lost, and divides with focus collagen and soft
Osteocyte is lost.3=Toluidine blue stainings, moderate cartilage is lost, and is lost with the division of multifocal collagen and cartilage cell.4=
Toluidine blue staining, hence it is evident that cartilage is lost, loses with the division of multifocal collagen and cartilage cell.5=Toluidine blue stainings, sternly
Weight amalgamation cartilage is lost, and is lost with the division of multifocal collagen and cartilage cell.
4. result
4.1 pairs of influences of sufficient pawl swelling degree
1) rat foot claw intracutaneous injection CFA (10g/L) 0.1ml, with sufficient pawl capacity measurer in cause inflammation after d12, d16,
D20, d24, d28 periodic detection rat foot claw swelling.Result shows：Sufficient pawl swelling degree periodic detection value model group is more normal
Control group substantially increases (P ＜ 0.05).
2) the 3rd day (d3), d7, d11 measure sufficient pawl volume to positive control drug prednisone 10mg/kg gastric infusions (ig) afterwards,
Compare with model group, sufficient pawl swelling degree substantially mitigates (P ＜ 0.05~0.01).
3) d3, d7, d11 measure sufficient pawl volume to Paeoniflorin tolysulfonyl compound 100mg/kg gastric infusions (ig) afterwards, with
Model group compares, it is seen that sufficient pawl swelling degree substantially mitigates (P ＜ 0.05~0.01)
4.2. to the influence of articular index
1) rat foot claw intracutaneous injection CFA (10g/L) 0.1ml, scores by 0~4 Pyatyi, fixed in d3, d7, d11 after administration
Phase assesses articular index.Result shows：Articular index assessment integral model group substantially increases (P ＜ compared with Normal group
0.01)。
2) d3, d7, d11 assess articular index to positive control drug prednisone 10mg/kg gastric infusions (ig) afterwards, with model group
Compare, articular index assessment integration is significantly reduced (P ＜ 0.05~0.01).
3) d3, d7, d11 assess articular index to Paeoniflorin tolysulfonyl compound 100mg/kg gastric infusions (ig) afterwards, with
Model group compares, it is seen that can reduce articular index integration (P ＜ 0.05~0.01).
4.3. to the influence of thymus index and spleen index
1) rat foot claw intracutaneous injection CFA (10g/L) 0.1ml, anaesthetizes after d12 is administered and puts to death rat, weighs, and takes chest
Gland and spleen are weighed, and calculate Thymus and Spleen index.Result shows：Model group index and spleen index is significantly greater than Normal group
(P ＜ 0.01), but thymus index no significant difference.
2) positive control drug prednisone 10mg/kg gastric infusions (ig), compare thymus index and (P are obviously reduced with model group
＜ 0.05), index and spleen index is had no significant effect.
3) Paeoniflorin tolysulfonyl compound 100mg/kg (ig) is and strong with model group than index and spleen index no significant difference
Loose group compare also no significant difference.
4) influence of each test medicine of overall merit to Thymus and Spleen index, positive control prednisone has significantly
Immune organ inhibitory action, can be obviously reduced thymus index, and the then suppression without immune organ of Paeoniflorin tolysulfonyl compound
Effect.
The influence of T and the B lymphocyte proliferation reaction for 4.4. inducing LPS and ConA
1) rat foot claw intracutaneous injection CFA (10g/L) 0.1ml, anaesthetizes after d12 is administered and puts to death rat, aseptic to take spleen
Lymphocyte suspension is prepared with RPMI1640, on 96 well culture plates, 100 μ l cell suspensions of every hole addition (final concentration of 5 ×
106·ml-1) and LPS (final concentration 4mgL-1) or ConA (final concentration 3mgL-1), whole volume is 200 μ l, respectively sets 3 again
Hole, puts 37 DEG C, 5%CO2Incubator culture 48h, induction T, B lymphocyte proliferation.Result shows：Model group and control group ratio
Compared with the either T of LPS or ConA inductions and B lymphocyte proliferation reaction is subject to substantially to suppress (P ＜ 0.01).
2) positive control drug prednisone 10mg/kg gastric infusions (ig), compare with model group, either LPS or ConA
T and the B lymphocyte proliferation reaction of induction are further subject to substantially to suppress (P ＜ 0.001).
3) T and B lymphocyte proliferation reaction that Paeoniflorin tolysulfonyl compound group is induced ConA have obvious enhancing
Effect, compares that there were significant differences (P ＜ 0.05~0.01) with model group, and then no significant difference is compared with prednisone group.Paeoniflorin
The T and B lymphocyte proliferation reaction that tolysulfonyl compound group is induced LPS have no significant effect, and compare with model group without substantially
Difference, but compare with prednisone group and have notable difference (P ＜ 0.01).
4.5 pairs of LPS induction peritoneal macrophage IL-1 β mRNA and TNF-α mRNA expression, Con A induction thymocyte lifes
Into the influence of IL-2mRNA expression
1) rat foot claw intracutaneous injection CFA (10g/L) 0.1ml, anaesthetizes after d12 is administered and puts to death rat, aseptic to take abdominal cavity
Macrophage culture, is expressed with LPS stimulating expression of macrophage IL-1 β mRNA and TNF-α mRNA, is determined with RT-PCR methods.It is another aseptic
Take rat chest gland RPMI1640 and prepare T lymphocyte suspension cultures, stimulate thymus T cells IL-2mRNA to express with ConA, use
RT-PCR methods are determined.Result shows：Model group compares with control group, LPS stimulating expression of macrophage IL-1 β mRNA and TNF-α mRNA
Expression is significantly improved (P ＜ 0.01), and ConA stimulates thymus T cells IL-2mRNA expression nothings to significantly improve.
2) positive control drug prednisone 10mg/kg gastric infusions (ig), compare with model group, can substantially suppress LPS stimulations
Macrophage IL-1 β mRNA and TNF-α mRNA express (P ＜ 0.01), stimulate ConA thymus T cells IL-2mRNA expression without bright
Development rings.
3) Paeoniflorin tolysulfonyl compound is stronger to suppression that TNF-α mRNA is expressed, the suppression to IL-1 β mRNA expression
Effect is weaker.
4.6. to whole body pathology：Changes of weight, the incidence and severity 1 of forelimb, ear and afterbody lesion) respectively at injection
Adjuvant causes d16 (before administration) after inflammation, cause d20 (administration d3) after inflammation, cause after inflammation d24 (administration d7) and cause d28 (d11 is administered) after inflammation
Weigh, calculate increased weight.Body weight before body weight-administration after increment (g)=administration.Result shows, model group and control group ratio
Compared with body weight is decreased obviously；Identical with model group, Paeoniflorin tolysulfonyl compound group body weight is also decreased obviously.
2) incidence and severity of observed and recorded forelimb, ear and afterbody lesion, except control group, model group and each by reagent
There is hind leg and be injected the different degrees of swelling in position in thing group, part occurs ulcer or festers, forelimb after inflammation is caused with the time
There is mild swelling, partial rat tail with ear and tubercle occurs, its incidence and severity are substantially coincident before administration.
After starting administration, Paeoniflorin tolysulfonyl compound group, positive control drug prednisone group whole body pathology it is further without occurring
Deterioration, partly start mitigate.
4.7. joint pathology inspection
1) d28 (before administration) after injection adjuvant causes inflammation, i.e., put to death rat after each study medication administration d12, takes metapedes scorching
Property ankle-joint carry out HE dyeing, observe the pathological change of inflamed joints tissue：According to the order of severity to synovial hyperplasia, inflammation, blood
Pipe screen is scored.Inflammatory score scores by 0~3 level Four, and 0=is normal；The less cell infiltration of 1=soft tissues；2=moderates are scorching
Cellular infiltration；The obvious cell infiltrations of 3=.Result shows：Model group synovial hyperplasia substantially, has substantial amounts of neutral grain thin in synovial membrane
Born of the same parents infiltrate and a small amount of pannus generation and slight cartilage destruction；Positive control drug prednisone can substantially mitigate AA inflammation,
Mitigate synovial hyperplasia, reduce neutrophil infiltration and pannus generation；Paeoniflorin tolysulfonyl compound group can substantially subtract
Light AA inflammation, mitigates synovial hyperplasia, reduces neutrophil infiltration and pannus generation.
2) cartilage destruction scoring：By 0~5 six grades of scorings, 0=is normal；1=Toluidine blue stainings, slight cartilage is lost, nothing
Obvious collagen division and cartilage cell lose.2=Toluidine blue stainings, slight cartilage is lost, with the division of focus collagen and
Cartilage cell loses.3=Toluidine blue stainings, moderate cartilage is lost, and is lost with the division of multifocal collagen and cartilage cell.4
=Toluidine blue staining, hence it is evident that cartilage is lost, loses with the division of multifocal collagen and cartilage cell.5=Toluidine blue stainings,
Serious amalgamation cartilage is lost, and is lost with the division of multifocal collagen and cartilage cell.Result shows：Compare with control group, mould
Type group cartilage destruction is not obvious, and the cartilage destruction of Paeoniflorin tolysulfonyl compound group is not also obvious.
Claims (3)
1. a kind of acylated glycosides compound, it is characterized in that it has following structural formula：
Formula one：
R1=H；R2=p-toluenesulfonyls.
2. the preparation method of the acylated glycosides compound described in claim 1, it is characterized in that it is made up of the following steps：
Step one,
R1=H
The dissolving of said structure glycoside in a solvent, the solution containing paratoluensulfonyl chloride is added, after stirring, fully reaction, is fitted
Amount water washing, organic solvent extraction, by extract anhydrous sodium sulfate drying, filtering is evaporated, and absolute ethyl alcohol recrystallization obtains powder
Last shape solid；
Step 2,
The pulverulent solids that step one is obtained eluent dissolves, and silica gel column chromatography is eluting, obtains pulverulent solids, as
Acylated glycosides compound described in claim 1.
3. application of the acylated glycosides compound described in claim 1 in the medicine for preparing various immunity diseases.
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