CN102895650B - Application of cyclotides ingredients in zizyphus jujube roots - Google Patents
Application of cyclotides ingredients in zizyphus jujube roots Download PDFInfo
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- CN102895650B CN102895650B CN201210443941.3A CN201210443941A CN102895650B CN 102895650 B CN102895650 B CN 102895650B CN 201210443941 A CN201210443941 A CN 201210443941A CN 102895650 B CN102895650 B CN 102895650B
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- discarine
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- cyclic peptide
- dipeptidyl peptidase
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Abstract
酸枣根中环肽类成分的用途，环肽类成分为Discarine-L和Discarine-M，此两个环肽类成分对二肽基肽酶-Ⅳ具有良好的抑制作用，并且对四氧嘧啶诱导的糖尿病小鼠的血糖有显著降低作用。Discarine-L和Discarine-M可作为二肽基肽酶-Ⅳ抑制剂用于制备治疗糖尿病的药物。The use of cyclic peptide components in jujube root. The cyclic peptide components are Discarine-L and Discarine-M. Diabetic mice had a significant reduction in blood sugar. Discarine-L and Discarine-M can be used as dipeptidyl peptidase-IV inhibitors for preparing medicines for treating diabetes.
Description
Technical field
[0001] the present invention relates to field of medicaments.
Background technology
[0002] dipeptidyl peptidase IV (DPP-IV) is the high specific serine protease existing with dimeric forms, and its natural substrate is glucagon-like-peptide-1 (GLP-1) and glucose pancreotropic hormone polypeptide (GIP).GLP-1 has different physiological roles, at pancreas, can increase the insulin secretion of dependence on the glucose, the secretion of inhibition glucagon makes beta Cell of islet hypertrophy; At gastrointestinal tract, can delay gastric emptying after the meal, thereby delay intestinal glucose absorption.GIP has insulin secretion accelerating function.GLP-1 and GIP in dipeptidyl peptidase IV (DPP-IV) energy fast degradation body, make it inactivation.If therefore medicine can suppress dipeptidyl peptidase IV, will increase GLP-1 and GIP level in body.Thereby performance increases insulin secretion, makes beta Cell of islet hypertrophy, increases the pharmacological action such as gastric emptying, delay glucose absorption after the meal.Therefore, DPP-IV inhibitor, by suppressing DPP-4 enzyme, can respond blood glucose and increase, and only promotes when being necessary the secretion of insulin, thereby reduction blood sugar level, to reduce hypoglycemic risk, and during treating with this medicine, can not increase patient's body weight, be described as the antidiabetic drug (Cheng Wei etc. of " clever ", clinical rational drug use, 2011,4(2 A): 145-146).
Ziziphi Spinosae (
ziziphus jujuba) have another name called sour jujube, sour jujube, wild Fructus Jujubae, Fructus Choerospondiatis, Pueraria lobota pin etc., be Rhamnaceae (Rhamnaceae) jujube.The nutritive value of Ziziphi Spinosae is very high, also has medical value, and it is used as medicine with kernel, has the effect (Zhou Chengming etc., 80 kinds of conventional Chinese herbal medicine are cultivated, extract, marketed (second edition): Chinese agriculture publishing house, 2008) of tonifying liver gallbladder, mind calming arresting sweating.People have carried out large quantity research to Ziziphi Spinosae chemical composition and pharmacologically active in recent years; find that it contains the compositions such as flavone, triterpene; there are the multiple pharmacological effect (Chen Wen etc. such as tranquilizing soporific, myocardial cell protection, arrhythmia, inhibition atherosclerosis; time precious traditional Chinese medical science traditional Chinese medicines; 2011,22(7): 1726-1728).But up to the present, have no Radix ziziphi mauritianae medium ring peptide constituents dipeptidyl peptidase-IV is suppressed to active report.
Summary of the invention
The object of this invention is to provide the application of Radix ziziphi mauritianae medium ring peptide constituents in the anti-dipeptidyl peptidase-IV medicine of preparation
.
the present invention is the purposes of Radix ziziphi mauritianae medium ring peptide constituents, and Radix ziziphi mauritianae medium ring peptide constituents is compound 1:discarine-L and compound 2:Discarine-M, the purposes in preparing dipeptidyl peptidase-IV inhibitors;
The present invention adopts enzyme linked immunosorbent assay (Miao Lei etc., Chinese Pharmacological Bulletin, 2009,25 (3): the anti-dipeptidyl peptidase-IV of 411-414) having studied Ziziphi Spinosae cyclic peptide components D iscarine-L and Discarine-M is active.And studied Discarine-L and the Discarine-M hypoglycemic activity to alloxan induction diabetic mice.Result shows, Discarine-L and Discarine-M are good dipeptidyl peptidase-IV inhibitors, and all can obviously reduce the blood glucose of diabetic mice.Cyclic peptide components D iscarine-L in Radix ziziphi mauritianae and Discarine-M with pharmaceutically acceptable carrier or other excipient are combined, according to conventional method, make interior injection or other dosage forms with type and parenteral administration of oral administration administration, can be for exploitation antidiabetic medicine.
The specific embodiment
The present invention is the purposes of Radix ziziphi mauritianae medium ring peptide constituents, and its cyclic peptide composition is compound
1for Discarine-L, compound 2 is Discarine-M, and its purposes is dipeptidyl peptidase-IV inhibitors.
Compound structure is as follows:
The purposes of Radix ziziphi mauritianae medium ring peptide constituents of the present invention is dipeptidyl peptidase-IV inhibitors.Dipeptidyl peptidase-IV inhibitors of the present invention refers to the medicine for the preparation for the treatment of diabetes.Above-mentioned cyclic peptide composition is combined with pharmaceutically acceptable carrier or other excipient, makes interior injection or other dosage forms with type and parenteral administration of oral administration administration according to conventional method.
Compound
1for Discarine-L, its Structural Identification is as follows:
White amorphous powder, MS:m/z 519.6[M+H]
+,
1h-NMR (400 MHz, CDCl
3) δ ppm:4.70(1H, m), 4.37(1H, m), 6.74(1H, d,
j=9.0Hz), 4.20(1H, m), 7.56(1H, d,
j=10.4Hz), 4.02(1H, m), 2.82(1H, m), 4.92(1H, d,
j=4.0Hz), 6.85(2H, m), 7.26(1H, m) and, 6.80(1H, m), 2.22(1H, m), 1.02(3H, d,
j=6.4Hz), 0.90(3H, d,
j=6.4Hz), 8.19(1H, d,
j=10.4Hz), 2.70(3H, d,
j=6.4Hz), 1.72(1H, m),
1.50（1H,?m），1.05（1H,?m），0.80（3H,?t，
J=6.4Hz），0.60（3H,?d，
J=6.4Hz），2.17（6H,?m），1.25（1H,?m），1.73（1H,?m），1.25（2H,?m），0.72（3H,?d，
J=6.4Hz），0.69（3H,?d，
J=6.4Hz）；
13C-NMR(125?MHz，CDCl
3)δppm：156.5（C），79.4（CH），55.6（CH），170.5（C=O），51.6（CH），169.8（C=O），48.4（CH
2），71.6（CH），135.5（C），114.6（CH），118.4（CH），127.8（CH），127.3（CH），28.6（CH），14.8（CH
3），20.8（CH
3），171.4（C=O），72.0（CH），33.3（CH），
25.8（CH
2），10.8（CH
3），15.8（CH
3），41.5（N-CH
3），41.5（N-CH
3），42.8（CH
2），24.6（CH），22.2（CH
3），24.5（CH
3）。Above spectroscopic data and document (D. F. Morel, et al, Phytochemistry, 1995,39 (2): the Discarine-L report 431-434) is consistent, therefore this compound identification is Discarine-L.
Compound
2for Discarine-M, its Structural Identification is as follows:
White amorphous powder, ESIMS:m/z 457.8[M+H]
+,
1h-NMR(400 MHz, DMSO-d6) δ ppm:8.46(1H, d, J=10.4 Hz), 7.60(1H, d, J=10.0 Hz), 7.15(1H, d, J=8.0 Hz), 7.10(1H, d, J=8.0 Hz), 7.01(1H, d, J=8.0Hz), 6.90(1H, d, J=8.0Hz), 6.74(1H, d, J=8.0 Hz), 6.64(1H, m), 6.50(1H, m), 6.40(1H, d, J=8.0 Hz), 5.86(1H, d, J=14.0 Hz), 4.90(1H, m), 4.57(1H, m)), 3.80(1H, m), 2.33(1H, m), 2.10(1H, m), 1.41(H, m), 1.34(1H, m), 1.28(1H, m), 1.10(3H, d, J=6.4 Hz), 0.98(3H, d, J=6.4Hz), 0.95(3H, d, J=6.4 Hz), 0.82(3H, d, J=6.4 Hz), 0.71(3H, d, J=6.4Hz), 0.48(3H, d, J=6.4Hz),
13C-NMR(125?MHz，DMSO-d6)δppm：171.4（C=O）168.0（C=O），164.1（C=O），155.8（C），149.7（CH），131.1（C），130.9（CH），130.3（CH），125.7?（CH），122.8（CH），121.4（CH），121.0（CH），116.8（CH），82.2（CH），54.9（CH），52.1（CH），38.6（CH
2），30.0（CH），28.2（CH），23.4（CH），23.1（CH
3），21.5（CH
3），21.4（CH
3），21.3（CH
3），20.4（CH
3），14.4（CH
3）。Above spectroscopic data and document (S. R. Giacomelli, et al, Phytochemistry, 2004, the Discarine-M report in 65:933-937) is consistent, therefore this compound identification is Discarine-M.
embodiment mono-: the separation of Radix ziziphi mauritianae medium ring peptide constituents of the present invention:
Take the powder 5kg of the root of dry Ziziphi Spinosae, add 95% ethanol 40L hot reflux to extract 4h, sucking filtration is dry, then adds 95% ethanol 20L hot reflux to extract 2h, then sucking filtration.The extracting solution merging of twice is concentrated into dry, obtains ethanol extraction extractum 1200g, and this extractum adds 10L distilled water and suspends, and uses the hydrochloric acid adjust pH of 2M to 2-3, sucking filtration after standing 15h.Filtrate, by 10L extracted with diethyl ether twice, merges organic facies, is evaporated to the dry acid ether extract (120g) that obtains, and water arrives 8-9 with liquid ammonia alkalinization to pH value, then uses 10L extracted with diethyl ether twice, merges organic facies, obtains alkaline ether extract (80g).The ether extract macroporous resin column chromatography of alkalescence, with methanol/water=0/1,20/80,40/60,60/40,80/20,1/0 eluting successively, the elution fraction of collection methanol/water=60/40, recrystallizing methanol obtains compound
1(Discarine-L) (800 mg), crystalline mother solution with MCI-Gel for column chromatography methanol/water=60/40 eluting obtain compound
2(Discarine-M) (550 mg).
embodiment bis-: enzyme linked immunosorbent assay is measured the anti-dipeptidyl peptidase-IV activity of cyclic peptide composition:
By Discarine-L, Discarine-M with press down two Copeptin A and be dissolved in respectively DMSO, being made into concentration is the sample liquid of 1mg/mL, and then using dipotassium hydrogen phosphate-potassium phosphate buffer (PH=8.2) dilution is the liquid to be measured of variable concentrations (1-50 μ g/mL).Getting 120 μ L dipotassium hydrogen phosphate-potassium phosphate buffers (PH=8.2) and 20 μ L sample liquid and 0.5U/mL DPPIV 10 μ L mixes, in 4 ℃, cultivate 20min, add 1 mmol/L substrate (glycyl proline paranitroanilinum dissolves with phosphate buffer), 50 μ L, in 37 ℃ of incubation 30min, under 405 nm wavelength, measure OD value, more than react on 96 enzyme mark orifice plates and complete, reaction cumulative volume is 200 μ l.Each test sample is done 3 multiple holes simultaneously, averages, and 3 repeated experiments.Press down two Copeptin A as the positive control of this law, set blank and negative control simultaneously.Calculate the suppression ratio of enzymatic activity: suppression ratio=[(complete suppressed group of OD unrestraint agent group-OD)-(at the bottom of OD sample-OD sample copy)]/(complete suppressed group of OD unrestraint agent group-OD) * 100%, by the suppression ratio calculation of half inhibitory concentration (IC of variable concentrations
50value).Wherein, blank group replaces sample solution with buffer; Inhibition group completely replaces sample solution with pressing down two Copeptin A (0.1 mg. mL-1).At the bottom of sample copy, group replaces enzyme by buffering.
Result is as table 1.Result shows that Discarine-L and Discarine-M have stronger dipeptidyl peptidase IV and suppress active, its IC
50value is respectively 16.3,20.5 μ g/mL, its effect crystallization positive drug.
Table 1. Discarin-L and the Discarin-M impact on dipeptidyl peptidase IV activity
| Sample | Suppression ratio is (during final concentration 100 μ g/mL, %) | IC 50Value (μ g/mL) |
| Discarine-L | 97.2±2.8 | 16.3±1.8 |
| Discarine-M | 95.4±2.1 | 20.5±2.0 |
| Press down two Copeptin A(positive drug) | 99.5±1.8 | 12.3±1.5 |
embodiment bis-: the experiment to the blood sugar influence of alloxan induction diabetic mice of cyclic peptide composition
150 of male mice in kunming, before experiment, fasting is 12 hours, chooses at random 10 as Normal group, all the other lumbar injection 200mg/kg alloxan, after 72 hours, survey blood glucose value, choosing wherein 80 blood glucose value >11.1mmol/L persons as diabetic mice.Diabetic mice is divided into high, medium and low three dosage group (40 mg/Kg of Compound D iscarine-L at random, 20 mg/Kg, 10 mg/Kg), high, medium and low three the dosage groups of Compound D iscarine-M (40 mg/Kg, 20 mg/Kg, 10 mg/Kg), model control group, glibenclamide positive controls (20mg/Kg), makes there was no significant difference between each blood glucose value mean of organizing, 10 every group.Each organizes gastric infusion or normal saline every day, and continuous 2 weeks, in last fasting, within 12 hours, after administration, within 1 hour, from mouse orbit, get blood again, with blood glucose examination bar, measure blood sugar content, the results are shown in Table 2.
Table 2. Discarin-L and the Discarin-M blood sugar influence to model induced by alloxan diabetic mice
| Group | Dosage (mg/kg) | Blood glucose value (mmol/L) |
| Normal group | — | 5.4± 0.8** |
| Model control group | — | 19.3 ± 2.6 |
| Glibenclamide group | 20 | 8.1 ± 1.9** |
| Discarine-L group (height) | 40 | 9.3 ± 1.8** |
| Discarine-L group (in) | 20 | 10.6 ± 2.3** |
| Discarine-L (low) | 10 | 13.4 ±2.8* |
| Discarine-M group (height) | 40 | 10.0 ± 2.0** |
| Discarine-M group (in) | 20 | 11.6 ± 2.3** |
| Discarine-M (low) | 10 | 14.4 ±2.8 |
Note: with model control group comparison, * p<0.05, * * p<0.01.
As shown in Table 2, high with Discarine-M, the middle dosage group of Discarine-L blood glucose is compared remarkable reduction (P<0.01) with model control group, and blood glucose declines and is dose-dependence, and the hypoglycemic effect of Discarine-L and Discarine-M high dose group and positive drug glibenclamide approach.
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|---|---|---|---|
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Non-Patent Citations (4)
| Title |
|---|
| ADEMIR FARIAS MOREL et al..CYCLOPEPTIDE ALKALOIDS OF DISCARIA FEBRIFUGA (RHAMNACEAE).《Phytochemistry》.1995,第39卷(第2期),第431-434页. |
| Cyclic peptide alkaloids from the bark of Discaria americana;S.R. Giacomelli et al.;《Phytochemistry》;20041231;第65卷;第933–937页 * |
| CYCLOPEPTIDE ALKALOIDS OF DISCARIA FEBRIFUGA (RHAMNACEAE);ADEMIR FARIAS MOREL et al.;《Phytochemistry》;19951231;第39卷(第2期);第431-434页 * |
| S.R. Giacomelli et al..Cyclic peptide alkaloids from the bark of Discaria americana.《Phytochemistry》.2004,第65卷第933–937页. |
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