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CN105497044A - Pharmaceutical composition for treatment of diseases or illness affected by neuronal injury - Google Patents

Pharmaceutical composition for treatment of diseases or illness affected by neuronal injury Download PDF

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CN105497044A
CN105497044A CN201510624256.4A CN201510624256A CN105497044A CN 105497044 A CN105497044 A CN 105497044A CN 201510624256 A CN201510624256 A CN 201510624256A CN 105497044 A CN105497044 A CN 105497044A
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compound
pharmaceutical composition
ethanol
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disease
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朱正直
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Abstract

The present invention relates to a pharmaceutical composition for the treatment of diseases or illness affected by neuronal injury, and the pharmaceutical composition comprises a therapeutically-effective amount of a compound (I), atropine as a neuroprotective agent and a pharmaceutically acceptable carrier. The compound (I) has a structure shown as (I), and can be obtained from dry whole plantain herb by extraction, separation and purification. In vitro tests show that the compound can resist A beta 25-35 induced PC12 neuronal apoptosis, and can be applied in the development of neuroprotective drugs.

Description

A kind of Pharmaceutical composition being used for the treatment of disease or the disease affected by neuronal damage
Technical field
The present invention relates to the Pharmaceutical composition being used for the treatment of disease or the disease affected by neuronal damage.
Background technology
Herba Plantaginis is the dry herb of Plantaginaceae Plantaginaceae plant Herba Plantaginis PlantagoasiaticaL. or Plantago depressa Willd PlantagodepressaWilld., is the conventional Chinese medicine that " Chinese Pharmacopoeia " (version in 2010) is recorded.At present, the Plantaginaceae found has 3 genus, about 200 kinds, distributes all over the world.China only produces Plantago Plantago, about 13 kinds.Except Herba Plantaginis and Plantago depressa Willd, the more Herba Plantaginis of research also has Big Semen Plantaginis PlantagomajorL. both at home and abroad.Herba Plantaginis is sweet in flavor and cold in property, has diuretic, heat clearing away, improving eyesight, the effect of eliminating the phlegm.Cure mainly gonorrhea, hematuria, urinary obstruction, yellow cellulitis, edema, hematodiarrhoea, have loose bowels, conjunctival congestion and swelling pain, laryngalgia etc." grass opinion " carries " control hematuria, energy tonifying five ZANG-organs, improving eyesight, the little Herba Plantaginis of drying of profit just, leads to five types of stranguria "." book on Chinese herbal medicine meets former " carries " if empty spermatorrhea gas does not consolidate person's forbidding ".Nature and flavor and function sweet, cold, return the hands sun, Yangming Channel.Diuretic, heat clearing away, improving eyesight, eliminates the phlegm, and for urinary obstruction, stranguria with turbid discharge, leukorrhagia, hematuria, jaundice, edema, hematodiarrhoea is had loose bowels, epistaxis, conjunctival congestion and swelling pain, laryngalgia, cough, skin ulcer.
Till settled the present, from aforementioned 3 kinds of Herba Plantaginiss, separation andpreconcentration goes out more than 60 kinds of compounds, can be divided into the compositions such as iridoids, flavonoid, phenethyl glycoside, phenolic acids and fatty acid by its major structural types.Iridoids has diuresis, antibacterial, the liver poisoning that anti-Carbon tetrachloride causes, choleretic effect; Flavonoid can act on respiratory center, alleviates respiratory movement and antitussive, and excitosecretory nerve makes trachea and bronchial secretion increase and eliminate the phlegm; Phenethyl glycoside has antiinflammatory, antibacterial, the activity that suppresses Camp phosphodiesterase and aldose reductase; The functions such as phenolic acids has sterilization, it is white to rise, function of gallbladder promoting, anticoagulant.
Modern pharmacology research shows, Herba Plantaginis has multiple pharmacologically active, of many uses, may be used for treatment chronic bronchitis, acute icterohepatitis, gouty arthritis, hypertension, bacillary dysentery, latent nephritis, glaucoma and gout etc.
PC12 cell strain comes from pheochromocytoma, has neuronic feature, therefore as the research of neuronic model for the death pathways and mechanism of studying neuronal cell.Multinomial research shows, the cytotoxic effect of A β to PC12 cell is because oxidative stress, mitochondrial function exception etc. cause apoptosis.
Summary of the invention
Goal of the invention of the present invention is to provide a kind of Pharmaceutical composition being used for the treatment of disease or the disease affected by neuronal damage.Foregoing invention object of the present invention is achieved by technical scheme below:
Be used for the treatment of a Pharmaceutical composition for disease or the disease affected by neuronal damage, Pharmaceutical composition comprise treatment effective dose compound (I), as the atropine of neuroprotective and pharmaceutically acceptable carrier,
Further, described compound (1) is obtained by following methods:
A, Herba Plantaginis are pulverized, and extract with 85% alcohol heat reflux, merge extractive liquid, is concentrated into without alcohol taste, uses petroleum ether, ethyl acetate and water saturated n-butanol extraction successively, obtains petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively;
Acetic acid ethyl ester extract macroporous resin remove impurity in b, step a, uses 20% ethanol and 80% ethanol elution successively, collects 80% ethanol elution, and concentrating under reduced pressure obtains 80% ethanol elution thing extractum;
In c, step b, 80% ethanol elution extractum purification on normal-phase silica gel is separated, and obtains 5 components successively with the methylene chloride-methanol gradient elution that volume ratio is 90:1,60:1,30:1,15:1 and 1:1;
In d, step c, component 4 is separated further by purification on normal-phase silica gel, obtains 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 20:1,12:1 and 5:1;
In e, steps d, component 2 reverse phase silica gel of octadecylsilane bonding is separated, and be the methanol aqueous solution isocratic elution of 65% by concentration expressed in percentage by volume, collect 8-11 column volume eluent, eluent concentrating under reduced pressure obtains pure compound (I);
85% ethanol described in above-mentioned steps, 20% ethanol and 80% ethanol all refer to concentration expressed in percentage by volume.
As preferably, compound (I) and the atropinic weight ratio as neuroprotective are 3-10:1; Described compound (I) effective dose every day is 0.001-0.006g/kg human body.
As preferably, pharmaceutically acceptable carrier is selected from following a kind of or both above mixing: lactose, sucrose, gelatin, agar, pectin, Radix Acaciae senegalis, magnesium stearate, stearic acid, cellulosic lower alkyl ether, corn starch, potato starch, natural gum, fatty acid, glycerol monostearate.
Compound of the present invention, its pharmaceutically acceptable salt and containing they one of medical composition, there is following beneficial effect:
The invention provides a kind of medicine catalogue that can be used for preparing neuroprotective novel drugs, that has widened patient selects medicine scope, for the screening for the treatment of neuroprotective disease provides new active drug kind.At present, at application 20 μm of ol/LA β 25-35the rat effectively setting up In vitro culture, addicted in chromium tumor cell strain PC12 Apoptosis Model, utilizes compound of the present invention (I) to carry out controlled trial, proves that compound of the present invention (I) can partial agonistic A β 25-35the PC12 apoptosis of induction, proves that it has effective medical value preparing in neuroprotective medicine, can be used for being developed to the new medicine use of neuroprotective.And medical composition prepared by compound of the present invention or this compound also has same application, compound (I), its pharmaceutically acceptable salt, containing they medical composition finally all by oral or injection form be applied to need treatment patient.
Accompanying drawing explanation
Fig. 1 is compound (I) structural formula;
Fig. 2 is compound (I) two dimension 1h- 1hCOSY composes;
Fig. 3 is that MTT metabolic rate detects A β 25-35to the toxic action of PC12 cell;
Fig. 4 is A β 25-35on the impact of PC12 cell LDH release rate;
Fig. 5 is A β 25-35and/or compound (I) is on the impact of PC12 cell LDH release rate.
Detailed description of the invention
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 material, reagent source:
Ethanol, petroleum ether, ethyl acetate, n-butyl alcohol, dichloromethane are analytical pure, and purchased from Shanghai Ling Feng chemical reagent company limited, methanol, analytical pure, purchased from Jiangsu Han Bang chemical reagent company limited.
A β 25-35be purchased from sigma company of the U.S..MTT (nitroblue tetrazolium) is purchased from Amresco company of the U.S..LDH mensuration test kit is purchased from Nanjing and builds up biological company limited.Acridine orange (AO) is purchased from sigma company of the U.S..Ethidum Eremide (EB) is purchased from sigma company of the U.S..RNase enzyme is purchased from sigma company of the U.S..E.C. 3.4.21.64 is purchased from sigmaAnnexin company of the U.S..V-FITC cell apoptosis detection kit is purchased from Nanjing KaiJi Biology Science Development Co., Ltd.D-MEM/F12 culture medium dry powder is purchased from GibcoL company of the U.S..Horse serum is purchased from hycLon company of the U.S..Hyclone is purchased from Hangzhou Ilex purpurea Hassk.[I.chinensis Sims biological engineering company limited.Poly-D-lysine (PLL) is purchased from sigma company of the U.S..Compound (I) is made by oneself, and HPLC normalization purity is greater than 98%.
Instrument type: low-temperature and high-speed centrifuge, U.S. Heraeus; CO2 gas incubator, U.S. SHELL/JB; Superclean bench, Suzhou Decontamination Equipment Plant; The vigorous MK3 microplate reader of thunder, Finland Thermolabsystems; Fluorescence microscope, German Leica; Flow cytometer EpicsXL, CouLter company of the U.S.; Electrophoresis system, Liuyi Instruments Plant, Beijing; The vigorous MK3 microplate reader of thunder, Finland Thermolabsystems; Fluorescence microscope, German Leica; Flow cytometer EpicsXL, CouLter company of the U.S..
Embodiment 1: compound (I) is separated preparation and structural identification
A the dry herb (8kg) of () Herba Plantaginis is pulverized, (25L × 3 time) are extracted with 85% alcohol heat reflux, merge extractive liquid, be concentrated into without alcohol taste (6L), use petroleum ether (6L × 3 time), ethyl acetate (6L × 3 time) and water saturated n-butyl alcohol (6L × 3 time) to extract successively, obtain petroleum ether extract, acetic acid ethyl ester extract (333g) and n-butyl alcohol extract respectively; Acetic acid ethyl ester extract AB-8 type macroporous resin remove impurity in (b) step (a), use 20% ethanol (8L) and 80% (12L) ethanol elution successively, collect 80% ethanol elution, concentrating under reduced pressure obtains 80% ethanol elution thing extractum (152g); C in () step (b), 80% ethanol elution extractum purification on normal-phase silica gel is separated, successively with volume ratio be 90:1 (7 column volumes), the methylene chloride-methanol gradient elution of 60:1 (7 column volumes), 30:1 (8 column volumes), 15:1 (7 column volumes) and 1:1 (5 column volumes) obtains 5 components; D component 4 (38g) is separated further by purification on normal-phase silica gel in () step (c), successively with volume ratio be 20:1 (10 column volumes), the methylene chloride-methanol gradient elution of 12:1 (8 column volumes) and 5:1 (6 column volumes) obtains 3 components; E in () step (d), component 2 (11g) reverse phase silica gel of octadecylsilane bonding is separated, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 65%, collect 8-11 column volume eluent, eluent concentrating under reduced pressure obtains pure compound (I) (26mg).
Structural identification:
Colourless powder; HR-ESIMS shows [M+Na] +for m/z493.2604, can obtain molecular formula in conjunction with nuclear-magnetism feature is C 28h 38o 6, degree of unsaturation is 10.Hydrogen nuclear magnetic resonance modal data δ h(ppm, DMSO-d 6, 500MHz): H-2 (2.99, dd, J=18.8, 3.3), H-2 (2.49, dd, J=18.8, 2.2), H-3 (4.29, dd, J=3.3, 2.2), H-4 (3.69, d, J=9.4), H-6 (3.37, d, J=3.2), H-7 (2.14, dt, J=14.6, 3.5), H-7 (1.29, dd, J=14.6, 11.3), H-8 (1.62, m), H-9 (1.90, m), H-11 (1.18, m), H-11 (0.94, dd, J=13.2, 4.0), H-12 (1.88, m), H-12 (1.22, m), H-14 (1.05, m), H-15 (1.64, m), H-15 (1.17, m), H-16 (1.68, m), H-16 (1.36, m), H-17 (1.12, m), H-18 (0.66, s), H-19 (4.19, d, J=9.0), H-19 (3.86, d, J=9.0), H-20 (1.98, m), H-21 (0.97, d, J=6.7), H-22 (4.35, dt, J=13.3, 3.5), H-23 (2.42, brdd, J=17.0, 13.3), H-23 (1.92, m), H-27 (1.88, s), H-28 (1.94, s), 6-OH (3.16, d, J=9.4), carbon-13 nmr spectra data δ c(ppm, DMSO-d 6, 125Hz): 208.0 (C, 1-C), 42.8 (CH 2, 2-C), 74.3 (CH, 3-C), 69.7 (CH, 4-C), 60.8 (C, 5-C), 56.9 (CH, 6-C), 29.8 (CH 2, 7-C), 28.8 (CH, 8-C), 36.9 (CH, 9-C), 50.1 (C, 10-C), 22.1 (CH 2, 11-C), 39.3 (CH 2, 12-C), 43.2 (C, 13-C), 54.8 (CH, 14-C), 24.2 (CH 2, 15-C), 27.3 (CH 2, 16-C), 52.3 (CH, 17-C), 11.8 (CH 3, 18-C), 63.5 (CH 2, 19-C), 38.9 (CH, 20-C), 13.5 (CH 3, 21-C), 78.4 (CH, 22-C), 29.7 (CH 2, 23-C), 149.1 (C, 24-C), 122.1 (C, 25-C), 167.2 (C, 26-C), 12.6 (CH 3, 27-C), 20.7 (CH 3, 28-C), carbon atom labelling is shown in Fig. 1. 1h- 1hCOSY spectrum (Fig. 2) shows that compound (I) is containing-CH 2cH (OR)-part-structure [δ H2.99 (dd, J=18.8,3.3Hz, H-2 β), 2.49 (dd, J=18.8,2.2Hz, H-2 α), 4.29 (dd, J=3.3,2.2Hz, H-3)]; In HMBC spectrum, H-3 and C-1 (δ c208.0), H-2 β and C-4 (δ c, and H-3 and C-5 (δ 69.7) c60.8) dependency further demonstrate that said structure is inferred.These data show, compound (I) meets containing other circulus the requirement that degree of unsaturation is 10.Coupling in compound (I) between H-3 and H-4 shows there is about dihedral angle of 90 ° between the two.C-19 (δ in compound (I) c63.5), H-19 [δ h4.19 (d, J=9.0Hz), 3.86 (d, J=9.0Hz)] and H-3 (δ h4.29) chemical shift shows to there is an oxo bridge between C-3 and C-19.C-19 (δ in HMBC spectrum c63.5) with H-3 (δ h4.29) and C-3 (δ c74.3) with H-19 α (δ h4.19) the above-mentioned inference of relevance verification.Comprehensive HMBC, 1h- 1the two-dimensional spectrums such as HCOSY, ROESY and pertinent literature, determine this compound structure as shown in Figure 1.
The compound main uses of the present embodiment is the medicine for the preparation of neuroprotective.
Embodiment 2: compound (I) pharmacological action is tested
One, test method
1, cell culture
In the D-MEM/F12 culture fluid containing 10% horse serum, 5% hyclone, (37 DEG C, volume fraction is the CO of 0.05 to PC12 Growth of Cells 2, saturated humidity), routine passage.Culture vessel 0.005%PLL solution (molecular weight is 150,000-300,000) bag quilt during experiment, increases PC12 cell to culture vessel adhesive force.
2, A β 25-35condensed state process
A β 25-35be dissolved in aseptic double-distilled water, final concentration is 2.0mmol/L, subpackage ,-20 DEG C of preservations, before use, hatches 4-7d for 37 DEG C.
3, medicine is to the process of PC12 cell
The PC12 cell of exponential phase is inoculated in culture plate respectively by certain density, after its growth is stable, is divided into groups by cell, i.e. matched group, A β 25-35apoptosis-induced group and compound (I) interference A β 25-35apoptosis-induced group.Wherein A β 25-35apoptosis-induced group, add the A β of condensed state with culture fluid 25-35storage liquid, prepares certain density working solution, adds Tissue Culture Plate respectively, and every hole adds cell suspension more respectively, CO 2incubator is cultivated.Every group all establishes 3 parallel holes; Secondly according to 20 μm of ol/LA β 25-35, apoptosis group is interfered in setting compound (I), and cell with variable concentrations compound (I) pretreatment 1h, then gives final concentration 20 μm of ol/LA β respectively 25-35cultivate.
4, mtt assay measures cell survival rate
Take the logarithm the cell of trophophase with 2-5 × 10 5the initial concentration of/mL is inoculated in every hole 160 μ L in 96 well culture plates respectively, after its growth is stable, adds the A β of variable concentrations 25-35, cell controls group only adds equivalent culture fluid, and often group establishes three parallel holes.Cultivate 96 hours, every hole adds MTT solution (PBS preparation) the 20 μ L of 5mg/mL, and continue after mixing to cultivate 4h, abandon supernatant, every hole adds DMSO0.2mL, and vibration 10min, with blank control wells zeroing, measures each hole optical density value under 490nm wavelength.
5, the mensuration of LDH burst size
Be inoculated in the cell of 24 porocyte culture plates, after drug treating 48h, it is for subsequent use that supernatant is respectively organized in sucking-off, and each group cell adds 0.1%NP-401mL effect 10min, collects supernatant for subsequent use.Measure test kit according to LDH to illustrate, detect the LDH activity of each group of supernatant and 0.1%NP-40 respectively, LDH release rate=OD supernatant/ (OD supernatant+ OD 0.1%NP-40) × 100%.
6, flow cytometry apoptosis
By AnnexinVFITC test kit description operation:
(1) after cell process certain hour, the centrifugal 5min of 2000rpm/min, cell number 1-5 × 10 5;
(2) cell secondary is cleaned, the centrifugal 5min of 2000rpm with PBS;
(3) 500 μ LBindingbuffer suspension cells are added;
(4) after adding 2 μ LAnnexinV-FITC mixings, 5 μ LPropidiumIodide (PI) are added, mixing, room temperature, lucifuge reaction 20min;
(5) flow cytometer (BackmancoulterEpicsXL) detects, and excitation wavelength 488nm, utilizing emitted light 530nm., analyzes scatterplot.
7, statistical analysis
Result mean ± standard deviation represent, compare between two with t inspection, P<0.05 thinks statistical significance.
Two, result and conclusion
1, MTT measures A β 25-35on the impact of PC12 cytoactive
As can be seen from Figure 3, A β 25-35after process PC12 cell 4d, mtt assay detection display 20,40 μm of ol/LA β 25-35effect group light absorption value (A490) obviously reduces, and difference has statistical significance (P<0.05).Mtt assay detects and represents mitochondrial function, and result illustrates A β 25-35pC12 mitochondrial function is reduced, and viable count reduces, i.e. A β 25-35inhibitory action is had to the activity of PC12 cell, and relevant to activity.The results are shown in Table 1 and Fig. 3.
Table 1A β 25-35to the inhibitory action of PC12 Growth of Cells
Aβ 25-35(μmol/L) 0 10 20 40
Cell survival rate 100 87.04±4.3％ 73.4±3.2％ 68.6±3.08％
2, LDH burst size measures
10,20,40 μm of ol/LA β 25-35after effect PC12 cell 48h, by detecting the degree of injury of cell LDH release rate showed cell, result shows A β 25-35to the toxic action of PC12 cell, and relevant to the concentration of effect, 20,40 μm of ol/LA β 25-35effect group compares with matched group, and difference has statistical significance (Fig. 4).Select 20 μm of ol/LA β 25-35as damage group, observe the intervention effect of compound (I), effect 48h, result shows, 10ng/mL compound (I) can reduce PC12 cell LDH release rate, but difference is not remarkable; 100ng/mL, 1000ng/mL compound (I) group obviously reduces PC12 cell LDH release rate and [sees Fig. 5, number in the figure meaning: 1, control (matched group); 2,20 μm of ol/LA β 25-35; 3,20 μm of ol/LA β 25-35+ 1.0ng/ml compound (I); 4,20 μm of ol/LA β 25-35+ 10ng/ml compound (I); 5,20 μm of ol/LA β 25-35+ 100ng/ml compound (I); 6,20 μm of ol/LA β 25-35+ 1000ng/ml compound (I); Wherein 4,5,6 three groups and 20 μm of ol/LA β 25-35group compares p<0.05].
3, the flow cytometry that Annexin V-PI dyes detects apoptosis
Each group of PC12 cell flow cytometer after treatment, and scatterplot is analyzed (MuLticycL software processes), display matched group, 10 μm of ol/LA β 25-35, 20 μm of ol/LA β 25-35after effect 48h, the apoptosis rate of PC12 cell is respectively 2.1 ± 0.3%, and 9.3 ± 0.5% and 18.6 ± 3.4%, apoptosis rate and A β 25-35dosage becomes positive correlation, compare difference with matched group and there is significant, anticipate with 100ng/mL compound (I), apoptosis rate is reduced to 11.2 ± 3.7%, difference has statistical significance (to compare with matched group, *: P<0.05, * *: P<0.01; With 20 μm of ol/LA β 25-35group compares, #:P<0.05), display compound (I) anti-A β 25-35cause apoptotic effect.
Flow cytometry PC12 apoptosis rate ( n=3).Sum up, this research finds application 20 μm of ol/LA β 25-35effectively can set up the rat of In vitro culture addicted to chromium tumor cell strain PC12 Apoptosis Model; Compound (I) can partial agonistic A β 25-35the PC12 apoptosis of induction.
Embodiment 3
The preparation of tablet: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartaric acid or citric acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid, excipient is added, pelletizing press sheet than the ratio for 1:9 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 tartaric acid or citric acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric 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 tartaric acid or citric acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid, add excipient in itself and excipient weight than the ratio for 1:9, make capsule or granule.
Embodiment 6
The preparation of injection: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartaric acid or citric acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid, inject routinely with water, fine straining, injection 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 tartaric acid or citric acid or formic acid or ethanedioic acid etc., mineral acid example hydrochloric acid or sulphuric acid or phosphoric acid, be dissolved in sterile water for injection, stirring makes molten, filter with aseptic suction funnel, aseptic fine straining again, is sub-packed in ampoule, and after frozen drying, aseptic sealing by fusing obtains injectable powder.

Claims (4)

1. be used for the treatment of a Pharmaceutical composition for disease or the disease affected by neuronal damage, it is characterized in that: Pharmaceutical composition comprise treatment effective dose compound (I), as the atropine of neuroprotective and pharmaceutically acceptable carrier,
2. the Pharmaceutical composition being used for the treatment of disease or the disease affected by neuronal damage according to claim 1, is characterized in that, described compound (1) is obtained by following methods:
A, Herba Plantaginis are pulverized, and extract with 85% alcohol heat reflux, merge extractive liquid, is concentrated into without alcohol taste, uses petroleum ether, ethyl acetate and water saturated n-butanol extraction successively, obtains petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively;
Acetic acid ethyl ester extract macroporous resin remove impurity in b, step a, uses 20% ethanol and 80% ethanol elution successively, collects 80% ethanol elution, and concentrating under reduced pressure obtains 80% ethanol elution thing extractum;
In c, step b, 80% ethanol elution extractum purification on normal-phase silica gel is separated, and obtains 5 components successively with the methylene chloride-methanol gradient elution that volume ratio is 90:1,60:1,30:1,15:1 and 1:1;
In d, step c, component 4 is separated further by purification on normal-phase silica gel, obtains 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 20:1,12:1 and 5:1;
In e, steps d, component 2 reverse phase silica gel of octadecylsilane bonding is separated, and be the methanol aqueous solution isocratic elution of 65% by concentration expressed in percentage by volume, collect 8-11 column volume eluent, eluent concentrating under reduced pressure obtains pure compound (I);
85% ethanol described in above-mentioned steps, 20% ethanol and 80% ethanol all refer to concentration expressed in percentage by volume.
3. the Pharmaceutical composition being used for the treatment of disease or the disease affected by neuronal damage according to claim 1, is characterized in that: compound (I) and the atropinic weight ratio as neuroprotective are 3-10:1; Described compound (I) effective dose every day is 0.001-0.006g/kg human body.
4. the Pharmaceutical composition being used for the treatment of disease or the disease affected by neuronal damage according to claim 1, is characterized in that: pharmaceutically acceptable carrier is selected from following a kind of or both above mixing: lactose, sucrose, gelatin, agar, pectin, Radix Acaciae senegalis, magnesium stearate, stearic acid, cellulosic lower alkyl ether, corn starch, potato starch, natural gum, fatty acid, glycerol monostearate.
CN201510624256.4A 2015-09-25 2015-09-25 Pharmaceutical composition for treatment of diseases or illness affected by neuronal injury Withdrawn CN105497044A (en)

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Cited By (3)

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CN105111080A (en) * 2015-09-05 2015-12-02 林天样 Novel diterpene compound and medical application thereof
CN105153269A (en) * 2015-10-22 2015-12-16 淄博夸克医药技术有限公司 New triterpene compounds, and preparation method and medical application thereof
CN107778346A (en) * 2016-08-28 2018-03-09 成都宝科生物科技有限公司 A kind of highly oxidized noval chemical compound and its medical usage

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US20090088412A1 (en) * 2007-10-02 2009-04-02 Kaohsiung Medical University Composition for treating cancer cells and preparation method thereof
US20120196815A1 (en) * 2011-02-01 2012-08-02 Kansas University Center for Technology Commercialization Withanolide Isolated from Physalis longifolia and Analogs and Methods of Use Thereof

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105111080A (en) * 2015-09-05 2015-12-02 林天样 Novel diterpene compound and medical application thereof
CN105153269A (en) * 2015-10-22 2015-12-16 淄博夸克医药技术有限公司 New triterpene compounds, and preparation method and medical application thereof
CN107778346A (en) * 2016-08-28 2018-03-09 成都宝科生物科技有限公司 A kind of highly oxidized noval chemical compound and its medical usage

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Application publication date: 20160420