CN105348273A - Novel limonoid as well as preparation method and medical application thereof - Google Patents
Novel limonoid as well as preparation method and medical application thereof Download PDFInfo
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Abstract
The invention discloses a novel limonoid as well as a preparation method and medical application thereof, and belongs to the technical field of medicines. The novel limonoid is reported for the first time, is novel in structure and can be obtained by extraction, separation and purification of dry fructus aurantii immaturus. In-vitro tests show that the novel limonoid has a neuroprotection function on A beta(25-35)-induced hippocampal neuron injuries, also has the function of relieving A beta(25-35)-induced hippocampal neuron toxic injuries and can be used for developing neuroprotection medicines.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, be specifically related to from the dried immature fruit of citron orange of drying, be separated a kind of limonoid obtained, containing its pharmaceutical composition and its preparation method and application.
Background technology
The dried immature fruit of citron orange is clinical conventional Chinese medicine, is the dry young fruit of rutaceae bitter orange CitrusaurantiumL. and cultivated variety or sweet orange CitrussinensisOsbeck.5 ~ June collects the fruit from falling, and removing impurity, be two halves from middle part crosscut, dry or cryodrying, smaller directly dries or cryodrying.The place of production of the dried immature fruit of citron orange mainly contains the ground such as Sichuan, Jiangxi, Hunan, Fujian, produces bitter orange for famous-region drug with Jiangxi, and it is best in quality to produce the goose eye dried immature fruit of citron orange with Jiangxi.The dried immature fruit of citron orange has relieving stagnant Qi and disappears long-pending, effects such as loose ruffian of reducing phlegm.Clinically to stop in stagnant, the full distending pain of ruffian, heavy after rushing down dysentery, suffer from constipation, the stagnant vapour lock of phlegm, the obstruction of qi in the chest, accumulation of pathogens in chest, visceroptosis, in the prescription that " TCM Recipe voluminous dictionary " includes more than in the prescription of 7% containing the dried immature fruit of citron orange, it serves to show that Fructus Aurantii Immaturus is wide in clinical application.
The principle active component of the dried immature fruit of citron orange is volatile oil, alkaloids and flavones ingredient thereof.Wherein, volatile oil main component is d-limonen, Citric Acid etc.; Alkaloids composition is mainly synephrine, N-methyltyramine; Flavones ingredient is mainly naringin, Neohesperidin and hesperidine.The content difference of its synephrine of the former different dried immature fruit of citron orange of base, naringin and Hesperidin is very large.Containing Neohesperidin during the prematurity of bitter orange fruit, when fruit maturation, Neohesperidin disappears.Sweet orange fruit is not containing neohesperidin.The content of the Fructus Aurantii Immaturus that different growing stages is plucked, its effective constituent of Fructus Aurantii Immaturus of different-grain diameter size is also different.Composition in dried immature fruit of citron orange volatile oil is except outside the Pass having with kind, also relevant with the place of production.The content of synephrine is along with the prolongation of collecting period, and the content of synephrine reduces obviously, but its content difference and kind, the place of production do not have obvious dependency.Along with the increase of dried immature fruit of citron orange particle diameter, in the dried immature fruit of citron orange, the content of naringin is in rising trend.In the dried immature fruit of citron orange of Hunan, the content of total alkaloids and volatile oil is the highest, but with the Jiangxi dried immature fruit of citron orange, fructus ponciri trifoliatae no significant difference, sweet orange dried immature fruit of citron orange content is minimum.Wherein, the content of synephrine is the highest with the goose eye dried immature fruit of citron orange, and the content of Hesperidin is the highest with fructus ponciri trifoliatae, and the content of naringin is the highest with shaddock content.
Summary of the invention
The object of this invention is to provide and a kind ofly from the dried immature fruit of citron orange of drying, be separated a kind of limonoid obtained, containing its pharmaceutical composition and its preparation method and application.
Above-mentioned purpose of the present invention is achieved by technical scheme below:
There is the compound (I) of following structural formula:
The preparation method of described compound (I), comprise following operation steps: the dried immature fruit of citron orange of drying is pulverized by (a), extract with 70 ~ 80% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste, use sherwood oil, ethyl acetate and water saturated n-butanol extraction successively, obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; Acetic acid ethyl ester extract macroporous resin removal of impurities in (b) step (a), first use 5% ethanol elution, 6 column volumes, then use 70% ethanol elution, 8 column volumes, collect 70% elutriant, concentrating under reduced pressure obtains 70% ethanol elution enriched material; C in () step (b), 70% ethanol elution enriched material purification on normal-phase silica gel is separated, obtain 4 components successively with the methylene chloride-methanol gradient elution that volume ratio is 50:1,30:1,15:1 and 5:1; D in () step (c), component 4 is separated further by purification on normal-phase silica gel, obtain 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 8:1,5:1 and 2:1; E in () step (d), component 2 reverse phase silica gel of octadecylsilane bonding is separated, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 70%, collect 8 ~ 12 column volume elutriants, elutriant concentrating under reduced pressure obtains pure compound (I).
Further, in step (a), extract with 75% alcohol heat reflux, united extraction liquid.
Further, described macroporous resin is AB-8 type macroporous adsorbent resin.
A kind of pharmaceutical composition, the described compound (I) wherein containing treatment significant quantity and pharmaceutically acceptable carrier.
Described compound (I) application in the medicine preparing neuroprotective.
The application of described pharmaceutical composition in the medicine preparing neuroprotective.
When the compounds of this invention is used as medicine, directly can uses, or use with the form of pharmaceutical composition.
This pharmaceutical composition contains the compounds of this invention (I) for the treatment of significant quantity, and all the other are acceptable on pharmacology, nontoxic to humans and animals and pharmaceutically acceptable carrier of inertia and/or vehicle.
Described pharmaceutically acceptable carrier or vehicle are that one or more are selected from solid, semisolid and liquid diluent, filler and pharmaceutical preparation assistant agent.Pharmaceutical composition of the present invention is used with the form of per weight dose.Medicine of the present invention is applied to by form that is oral or injection the patient needing treatment.For time oral, tablet, slow releasing tablet, controlled release tablet, capsule, dripping pill, micropill, suspensoid, emulsion, powder or granule, oral liquid etc. can be made into; During for injecting, can be made into water-based or oily solution, aseptic powder injection, liposome or the emulsion etc. of sterilizing.
Accompanying drawing explanation
Fig. 1 is compound (I) structural formula;
Fig. 2 is that compound (I) calculates ECD and experiment ECD figure;
Fig. 3 is that compound (I) is on the impact of cultivating different time Hippocampal Neuron Cells vigor;
Fig. 4 is that compound (I) induces the impact of Hippocampal Neuron Cells vigor to A β 25-35;
Fig. 5 is the impact of compound (I) on the Apoptosis of Hippocampal Neurons that A β 25-35 induces.
Embodiment
Further illustrate essentiality content of the present invention below in conjunction with embodiment, but do not limit scope with this.Although be explained in detail the present invention with reference to preferred embodiment, those of ordinary skill in the art should be appreciated that and can modify to technical scheme of the present invention or equivalent replacement, and does not depart from essence and the scope of technical solution of the present invention.
Main raw, reagent source and instrument type:
Ethanol, sherwood oil, ethyl acetate, propyl carbinol, methylene dichloride are analytical pure, and purchased from Shanghai Ling Feng chemical reagent company limited, methyl alcohol, analytical pure, purchased from Jiangsu Han Bang chemical reagent company limited.
Through identifying that the dried immature fruit of citron orange is the dry young fruit of rutaceae bitter orange CitrusaurantiumL..
Embodiment 1: compound (I) is separated preparation and structural identification
A the dried immature fruit of citron orange (8kg) of drying is pulverized by (), (30L × 3 time) are extracted with 75% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste (6L), use sherwood oil (6L × 3 time), ethyl acetate (6L × 3 time) and water saturated propyl carbinol (6L × 3 time) to extract successively, obtain petroleum ether extract, acetic acid ethyl ester extract (375g) and n-butyl alcohol extract respectively; Acetic acid ethyl ester extract AB-8 macroporous resin removal of impurities in (b) step (a), first use 5% ethanol elution, 6 column volumes, use 70% ethanol elution, 8 column volumes again, collect 70% elutriant, concentrating under reduced pressure obtains 70% ethanol elution enriched material (129g); C in () step (b), 70% ethanol elution enriched material purification on normal-phase silica gel is separated, successively with volume ratio be 50:1 (8 column volumes), the methylene chloride-methanol gradient elution of 30:1 (8 column volumes), 15:1 (8 column volumes) and 5:1 (10 column volumes) obtains 4 components; D component 4 (27g) is separated further by purification on normal-phase silica gel in () step (c), successively with volume ratio be 8:1 (8 column volumes), the methylene chloride-methanol gradient elution of 5:1 (10 column volumes) and 2:1 (8 column volumes) obtains 3 components; E in () step (d), component 2 (15g) reverse phase silica gel of octadecylsilane bonding is separated, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 70%, collect 8 ~ 12 column volume elutriants, elutriant concentrating under reduced pressure obtains pure compound (I) (138mg).
Structural identification: HR-ESIMS shows [M+Na]
+for m/z607.2208, can obtain molecular formula in conjunction with nuclear-magnetism feature is C
31h
36o
11, degree of unsaturation is 14.Hydrogen nuclear magnetic resonance modal data δ
h(ppm, DMSO-d
6, 400MHz): H-3 (5.07, s), H-5 (3.15, dd, J=7.6, 5.4), H-6 (2.37, dd, J=16.5, 5.4), H-6 (2.27, dd, J=16.5, 7.6), H-11 (3.16, m), H-12 (1.91, d, J=15.5), H-12 (2.33, d, J=15.5), H-14 (1.59, dd, J=12.5, 6.8), H-15 (2.71, dd, J=18.6, 6.8), H-15 (3.54, dd, J=18.6, 12.5), H-17 (4.92, s), H-18 (1.01, s), H-19 (0.91, s), H-21 (7.24, s), H-22 (6.12, m), H-23 (7.32, t, J=1.7), H-28 (0.85, s), H-29 (0.81, s), H-30 (3.36, s), H-2 ' (5.92, dq, J=15.5, 1.7), H-3 ' (7.13, dq, J=15.5, 7.0), H-4 ' (1.93, dd, J=7.0, 1.7), 7-OCH
3(3.65, s), carbon-13 nmr spectra data δ
c(ppm, DMSO-d
6, 100MHz): 208.1 (C, 1-C), 77.5 (C, 2-C), 83.8 (CH, 3-C), 38.6 (C, 4-C), 42.3 (CH, 5-C), 31.1 (CH
2, 6-C), 173.0 (C, 7-C), 61.2 (C, 8-C), 61.7 (C, 9-C), 49.8 (C, 10-C), 55.9 (CH, 11-C), 27.0 (CH
2, 12-C), 33.6 (C, 13-C), 37.9 (CH, 14-C), 28.2 (CH
2, 15-C), 168.7 (C, 16-C), 82.2 (CH, 17-C), 22.6 (CH
3, 18-C), 17.1 (CH
3, 19-C), 120.2 (C, 20-C), 140.3 (CH, 21-C), 108.9 (CH, 22-C), 143.1 (CH, 23-C), 23.2 (CH
3, 28-C), 20.1 (CH
3, 29-C), 64.2 (CH, 30-C), 164.5 (C, 1 '-C), 121.3 (CH, 2 '-C), 148.0 (CH, 3 '-C), 17.7 (CH
3, 4 '-C), 52.1 (CH
3, 7-OCH
3), carbon atom mark is see Fig. 1.Infrared spectra shows this compound and contains hydroxyl and carbonyl group (3446cm
-1and 1736cm
-1).
13cNMR and DEPT spectrum shows 31 carbon signals altogether, comprises six methyl (methoxyl group), three methylene radical, 11 methynes (five alkene carbon, three containing oxygen carbon), and 11 quaternary carbon (four carbonyl carbon, an alkene carbon, three containing oxygen carbon).
1h and
13cNMR data analysis shows that this compound contains typical β substituted furan ring (δ H7.24, a s; 6.12, m and 7.32, t, J=1.7Hz; δ C120.2,140.3,108.9 and 143.1), a ketone carbonyl (δ C208.1) and three ester carbonyl group (δ C173.0,168.7 and 164.5) structures.Above-mentioned nuclear magnetic data shows that this compound is limonoid.In HMBC spectrum, H
3with the dependency of C-1 (δ C208.1) ,-19 (δ H0.91, s) and H-30 (δ H3.36, s) show that C-1 is ketone carbonyl.Two groups containing oxygen carbon signal (δ C77.5,2-C and 64.2,30-C; δ C61.7,9-C and 55.9,11-C) and corresponding proton signal (δ H3.36, s, H-30; δ H3.16, m, H-11) show that this compound contains 30,2-epoxide groups and 9,11-epoxide group.In addition, C-3 (δ C83.8) position is connected with a 2-butylene acetoacetic ester.In HMBC spectrum, H-3 (δ H5.07, s), H-2 ' (δ H5.92, dq, J=15.5,1.7Hz) and H-3 ' (δ H7.13, dq, J=15.5,7.0Hz) and the above-mentioned inference of relevance verification of C-1 ' (δ C164.5).Coupling constant (J=15.5Hz) between H-2 ' and H-3 ' shows that 2-butylene acetoacetic ester fragment is E.In HMBC spectrum ,-OCH
3(δ H3.65, s) and C-7 (δ C173.0), H-6 (δ H2.37, dd, J=16.5,5.4Hz; 2.27, dd, J=16.5,7.6Hz) show that C-6 (δ C31.1) is connected with a methoxycarbonyl fragment with the dependency of C-5 (δ C42.3) and C-7.According to the dependency of H-17 and C-20, C-21 and C-22 in HMBC spectrum, deducibility furan nucleus is positioned on C-17 position.In ROESY spectrum, H-5 and H
3-29, H-5 and H-17, and H-12 β and H-17 shows that these protons are beta configuration; H-14 and H
3the intersection peak of-18 shows that Me-18 and H-14 is α-configuration.Comprehensive hydrogen spectrum, carbon spectrum, HMBC spectrum and ROESY spectrum, and document is about correlation type nuclear magnetic data, can substantially determine this compound as shown in Figure 1, steric configuration is determined further by ECD test, theoretical value and experimental value basically identical (Fig. 2).
Embodiment 2: compound (I) pharmacological action is tested
One, material and instrument
Healthy female SD rat is provided by Shanghai The 2nd Army Medical College Experimental Animal Center.Compound (I) preparation method is shown in that embodiment 1, HPLC normalization method purity is greater than 98%.Dimethyl sulfoxide (DMSO), Semen Ginkgo extrac (EGb761, positive drug), MTT, A β 25-35, L-poly-lysine are all purchased from SIGMA company of the U.S..DMEM high glucose medium, Neurobasal, B27 are purchased from GIBCO company of the U.S..NSE is purchased from ABCOM company of Britain.Tunel is purchased from Wuhan Boster company.LDH test kit is purchased from Nanjing and builds up biochemical reagents company.
Electronic balance, Beijing Sai Duolisi.1815TCCO
2constant-temperature incubation case (Shel-Lab company of the U.S.), TH-2C isothermal vibration device (granary, Jiangsu experimental installation factory), dissecting microscope (Japanese OLYMPUS company), desk centrifuge (German Heraeus company), microplate reader (Japanese DynaTtech company), J2-HS completely automated high speed refrigerated centrifuge (Beckman company of the U.S.).
Two, test method
1, the original cuiture of hippocampal neurons
The detailed process that 1.1 hippocampal tissues are drawn materials
(1) the Healthy female SD rat general anaesthesia in pregnant for 16-18d age is sterilized, take out tire mouse after dissecting, put into D-Hank ' s balanced salt solution.
(2) slowly peel off skin of head and the skull of tire mouse under being positioned over dissecting microscope, then see the brain bilateral hemisphere of exposure.
(3) in hemicerebrum outer side careful separation meninx and pallium, i.e. visible hippocampal tissue.
(4) by the careful gripping hippocampal tissue of dissecting forceps, to be put in D-Hank ' s balanced salt solution rinsing repeatedly three times, thoroughly to be removed meninx and vascular surface.
(5) be separated the trace of blood and meninx, obtain hippocampal tissue, for the original cuiture of hippocampal neurons.
The detailed process of 1.2 hippocampal neurons original cuiture
(1) acquired hippocampal tissue eye scissors is fully shredded.
(2) by concentration be 0.125% trysinization 25min, then with containing 15% foetal calf serum DMEM high glucose medium complete culture solution stop digestion.
(3) in whizzer with the centrifugation 10min of 800rpm.
(4) supernatant discarded after adding substratum (NeurobasalMedium/B27), blown and beaten slowly by the elbow straw of flame polish with tip, piping and druming is until after piping and druming evenly repeatedly, and leave standstill 5min, the upper liquid of gained is required hippocampal neurons suspension.
(5) with tally counting, implant respectively by required cell density and wrap in advance by 96 orifice plates of L-poly-lysine or 6 well culture plates.Wherein every hole about 100 μ L in 96 well culture plates, in 6 well culture plates, every hole is about 1.5mL.
(6) CO of 37 DEG C is put into
2in incubator, for testing after cultivating 3d.
2, the screening of compound (I) optimum effect time and the best use of concentration
Design following seven component groups: 1. 10 μ g/mL groups; 2. 20 μ g/mL groups; 3. 40 μ g/mL groups; 4. 60 μ g/mL groups; 5. 80 μ g/mL groups; 6. 100 μ g/mL groups; 7. 0 μ g/mL group (i.e. blank group).Compound (I) each dosage component is not acted on 24h, 48h and 72h, every hole adds dimethyl sulfoxide (DMSO) 100 μ L respectively, after abundant dissolving, observe under the full-automatic microplate reader that wavelength is 570nm and measure its optical density value (OD value), getting the drug level of optical density(OD) highest point and the action time of the correspondence the best use of condition as the compound (I) of this experiment screening.MTT colorimetry is adopted to carry out the screening of the best use of condition.
3, compound (I) is on the neuronic impact of hippocampus in A β 25-35 induced damage situation
The A β 25-35 having prepared polymerase 17 d at 37 DEG C is adopted to set up the model of hippocampal neurons damage.Experiment is grouped as follows: A: blank group Control group, B:A β group, C: compound (I)+A β group, D: compound (I)+I (inhibitor)+A β group (adding the antagonist K252a of BDNF on compound (I)+A β group basis), E:EGB+A β group, F: compound (I) group, G:EGB group; After hippocampal neurons original cuiture 3d, in nutrient solution, add the A β 25-35 of 25 μ g/mL, again according to grouping administration respectively after 4h.
3.1 compounds (I) induce the impact of Hippocampal Neuron Cells toxicity to A β 25-35
3.1.1MTT the step of colorimetric determination hippocampal neurons vigor
(1) by after hippocampal neurons original cuiture 3d, compound (I) is added respectively: what does not add for 40 μ g/mL, EGb761:150 μ g/mL and blank group.MTT colorimetry is carried out after 48h.
(2) in 96 orifice plates, every hole adds the MTT10 μ L prepared respectively.
(3) CO of 37 DEG C is placed on
24h is hatched in incubator.
(4) after slowly sucking supernatant, every hole adds the dimethyl sulfoxide (DMSO) effect 10min of 100 μ L, makes it abundant dissolving.
(5) optical density value in every hole is measured under the full-automatic microplate reader being 570nm at wavelength.
3.2 compounds (I) are on the impact of the Apoptosis of Hippocampal Neurons that A β 25-35 induces
3.2.1 grouping is tested
By detecting the activity of the key protein enzyme Caspase-3 in Apoptosis of Hippocampal Neurons process, observe compound (I) induces Apoptosis of Hippocampal Neurons impact on A β 25-35.Experiment is divided into seven groups: A:Control group, B:A β group, C: compound (I)+A β group, D: compound (I)+I+A β group, E:EGB+A β group, F: compound (I) group, G:EGB group.
3.2.2 experimental procedure
(1), after giving medicine 48h respectively by grouping, careful collection cell also with after the centrifugal 5min of the rotating speed of 400rpm, is inhaled and is abandoned supernatant.
(2) hippocampal neurons is resuspended in lysis buffer, is positioned over and hatches 10min on ice.
(3) 4 DEG C of centrifuge, cell debris is removed, and collects supernatant (endochylema extracting solution) and is transferred in new centrifuge tube.
(4) centrifuge tube is put on ice, slowly adds the reaction buffer containing dithiothreitol (DTT).
(5) add caspase-3 substrate 1mM in adding to each centrifuge tube, in 37 DEG C of water-baths, hatch 60min.
(6) be positioned in microplate reader by 96 orifice plates, with distilled water zeroing, adjustment determined wavelength is 405nm, instrumental analysis recording light density value.
4, statistical analysis
Statistical analysis adopts SPSS10.0 software package, and experimental result represents with mean ± standard deviation (x ± s), and experimental data adopts variance analysis and Fisher'sPLSD inspection.With P≤0.05 for difference has significance statistical significance.
Three, result and conclusion
1, the original cuiture of hippocampal neuron
Observe the hippocampal neurons of cultivating, find that hippocampal neurons starts adherent in time inoculating 6-12h, cell process growth obviously, when hippocampal neurons grows into 3d, the growth conditions of cell paste is best, and the degeneration and the cavity that have no neurocyte are formed.
2, the screening (MTT colorimetry) of compound (I) optimum effect time and the best use of concentration
Compound (I) each dosage component is not acted on 24h, 48h and 72h, observes the change of its optical density(OD), get drug level corresponding to optical density(OD) highest point and time as the best use of concentration of the compound (I) of screening and Effect time.
As shown in Figure 3, the cell viability that 48h respectively organizes hippocampal neuron is obviously better than each group of 24h and 72h.The cell viability that 24h, 48h and 72h respectively organize hippocampal neuron all with the cell viability of 40 μ g/mL groups for the best.So compound (I) the best use of concentration filtered out is 40 μ g/mL, and the optimum effect time is 48h.
3, compound (I) is on the neuronic impact of hippocampus in A β 25-35 induced damage situation
3.1 compounds (I) induce the impact of Hippocampal Neuron Cells toxicity to A β 25-35
Each group of medicine acts on after 48h respectively, observes compound (I) to the impact of the Hippocampal Neuron Cells vigor that A β 25-35 induces.
As shown in Figure 4, after giving A β 25-35 modeling, A β group Hippocampal Neuron Cells vigor more blank group of cell viability obviously declines (P < 0.01), and this illustrates nerve injury model modeling success; By finding the comparison respectively giving A β 25-35 group, compound (I)+A β group comparatively A β group cell viability significantly increases (P < 0.01); Compound (I)+A β group compares with EGB+A β group without obvious statistical significance (P > 0.05); After adding antagonist, cell viability is (P < 0.05) between compound (I)+A β group and A β group; This illustrates that compound (I) can alleviate the neurotoxicity of A β 25-35 induction.
3.2 compounds (I) are on the impact of the apoptosis of hippocampus neurons that A β 25-35 induces
When the hippocampal neurons damage that A β 25-35 induces, caspase-3 activity is activated.OD value is higher shows that Apoptosis of Hippocampal Neurons degree is higher.Experimental result shows, and when hippocampal neurons is impaired, caspase-3 activity is activated, so caspase-3 activity can significantly increase, apoptotic degree increases.Visible in Fig. 5, the comparatively active obviously increase (P<0.01) of blank group caspase-3 of A β group, this illustrates nerve injury model modeling success; And the comparatively active significantly decline (P<0.01) of A β group extracellular caspase-3 of compound (I)+A β group; After adding antagonist, the activity of caspase-3 is between compound (I)+A β group and A β group (P<0.05); Compound (I)+A β group and EGB+A β group difference not obvious (P ﹥ 0.05); This illustrates the apoptotic effect that compound (I) has significant anti-A β 25-35 to induce to the hippocampal neuron under degree of impairment.
This is tested many levels from different perspectives and has inquired into compound (I) induces tire mouse hippocampal neurons injury neuroprotective to A β 25-35.First find when compound (I) concentration is 40 μ g/mL and action time is 48h, hippocampal neurons morphology and cell viability the best, carried out MTT colorimetry and found that compound (I) has the effect of growth promoting effects to hippocampal neurons under normal circumstances; Then, detect discovery compound (I) by cell viability and also there is the effect alleviating A β 25-35 induction Hippocampal Neuron Cells toxic damages.Then, by detecting the apoptotic effect that also demonstrate that compound (I) has significant anti-A β 25-35 to induce to the hippocampal neuron under degree of impairment to apoptosis-related protein enzyme.The effect of compound (I) is similar to positive drug Ginkgo biloda extract EGb 761.
Embodiment 3
The preparation of tablet: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, vehicle is added, pelletizing press sheet than the ratio for 1:7 in itself and excipient weight.
Embodiment 4
The preparation of oral liquid: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, oral liquid method for making makes oral liquid routinely.
Embodiment 5
The preparation of capsule or granule: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, add vehicle in itself and excipient weight than the ratio for 1:9, make capsule or granule.
Embodiment 6
The preparation of injection liquid: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, inject with water routinely, essence filter, injection liquid is made in embedding sterilizing.
Embodiment 7
The preparation of aseptic powder injection: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, be dissolved in sterile water for injection, stirring makes molten, filter with aseptic suction funnel, aseptic essence filter again, be sub-packed in ampoule, after frozen drying, aseptic sealing by fusing obtains powder injection.
The effect of above-described embodiment is essentiality content of the present invention is described, but does not limit protection scope of the present invention with this.Those of ordinary skill in the art should be appreciated that and can modify to technical scheme of the present invention or equivalent replacement, and does not depart from essence and the protection domain of technical solution of the present invention.
Claims (7)
1. there is the compound (I) of following structural formula:
2. the preparation method of compound according to claim 1 (I), it is characterized in that comprising following operation steps: the dried immature fruit of citron orange of drying is pulverized by (a), extract with 70 ~ 80% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste, use sherwood oil, ethyl acetate and water saturated n-butanol extraction successively, obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; B in () step (a), the removal of impurities of thing macroporous resin got by propyl carbinol, first use 5% ethanol elution, 6 column volumes, then use 70% ethanol elution, 8 column volumes, collect 70% elutriant, concentrating under reduced pressure obtains 70% ethanol elution enriched material; C in () step (b), 70% ethanol elution enriched material purification on normal-phase silica gel is separated, obtain 4 components successively with the methylene chloride-methanol gradient elution that volume ratio is 50:1,30:1,15:1 and 5:1; D in () step (c), component 4 is separated further by purification on normal-phase silica gel, obtain 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 8:1,5:1 and 2:1; E in () step (d), component 2 reverse phase silica gel of octadecylsilane bonding is separated, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 70%, collect 8 ~ 12 column volume elutriants, elutriant concentrating under reduced pressure obtains pure compound (I).
3. the preparation method of compound according to claim 2 (I), is characterized in that: in step (a), extracts, united extraction liquid with 75% alcohol heat reflux.
4. the preparation method of compound according to claim 2 (I), is characterized in that: described macroporous resin is AB-8 type macroporous adsorbent resin.
5. a pharmaceutical composition, is characterized in that: the compound according to claim 1 (I) wherein containing treatment significant quantity and pharmaceutically acceptable carrier.
6. compound according to claim 1 (I) application in the medicine preparing neuroprotective.
7. the application of pharmaceutical composition according to claim 5 in the medicine preparing neuroprotective.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105294816A (en) * | 2015-11-25 | 2016-02-03 | 吕涛 | New withanolide compound and preparation method thereof, and medical uses of new withanolide compound in neuroprotection |
| CN105461782A (en) * | 2016-01-14 | 2016-04-06 | 郑平珍 | Novel triterpene compound and preparation and medical application thereof |
| CN106146528A (en) * | 2016-07-15 | 2016-11-23 | 郑巧丹 | A kind of new diterpene alkaloid compounds and preparation method thereof and medical usage |
-
2015
- 2015-12-07 CN CN201510887943.5A patent/CN105348273A/en not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105294816A (en) * | 2015-11-25 | 2016-02-03 | 吕涛 | New withanolide compound and preparation method thereof, and medical uses of new withanolide compound in neuroprotection |
| CN105461782A (en) * | 2016-01-14 | 2016-04-06 | 郑平珍 | Novel triterpene compound and preparation and medical application thereof |
| CN106146528A (en) * | 2016-07-15 | 2016-11-23 | 郑巧丹 | A kind of new diterpene alkaloid compounds and preparation method thereof and medical usage |
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