CN106466310A

CN106466310A - Only son and heir's grass extract and preparation method thereof and the purposes in pharmacy

Info

Publication number: CN106466310A
Application number: CN201510502119.3A
Authority: CN
Inventors: 胡金锋; 熊娟; 武喜营; 刘新华; 潘礼龙; 杨国勋
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2015-08-17
Filing date: 2015-08-17
Publication date: 2017-03-01
Anticipated expiration: 2035-08-17
Also published as: CN106466310B

Abstract

The invention belongs to pharmaceutical technology field, it is related to only son and heir's grass extract and preparation method thereof and the purposes in pharmacy, the present invention extracts from rare plant only son and heir grass (Oresitrophe rupifraga Bunge) and separates the compound 1 obtaining formula 1 or formula 2 and compound 2, prove through pharmacological evaluation, this two compounds have significant neuroinflamation inhibitory activity, IC₅₀Value is respectively 7.21 and 9.39 μM, and all there is no toxic effect to cell survival, it is further useful for the medicine preparing prevention and/or treatment neurodegenerative diseases, in particular for treating Alzheimer (Alzheimer ' s disease,) and parkinson (Parkinson's disease, PD) AD.

Description

Only son and heir's grass extract and preparation method thereof and the purposes in pharmacy

Technical field

The invention belongs to pharmaceutical technology field, it is related to only son and heir's grass (Oresitrophe rupifraga Bunge) extract and its preparation side Method and the purposes in pharmacy, the especially purposes in preparation treatment neurodegenerative diseases medicine.

Background technology

Prior art discloses neurodegenerative diseases (Neuro-degenerative diseases) and include Alzheimer (Alzheimer's disease, AD), parkinson (Parkinson's disease, PD), amyotrophic lateral sclerosis (Amyotrophic lateral sclerosis, ALS) and Huntington Chorea (Huntington's disease, HD) etc..Research display, Described neurodegenerative diseases are in increase trend along with the aging of population in the world, and it not only has a strong impact on the life matter of patient Amount, and bring great financial burden to society.Although its pathogenesis indefinite at present, numerous studies data shows, Inflammation plays an important role in central nervous system degenerative disease.Studies have reported that, the ofneurodegenerative of inflammatory reaction mediation Pathological changes mainly lead to inflammatory factor such as nitric oxide (NO), COX-2 then by the overactivity of glial cell (cyclooxygenase-2) and tumor necrosis factor-alpha (tumor necrosis factor- α, TNF-α) etc. excessive discharge caused. Therefore, those skilled in the art concern about intracerebral neuroinflamation inhibitor research, develop further have treatment multiple The newtype drug of neurodegenerative diseases.

Research display, it is excellent that Chinese herbal medicine embodies multicomponent, Mutiple Targets, hypotoxic uniqueness in terms for the treatment of neurodegenerative diseases Gesture, such as classical prescription, proved recipe are clinically succeeded application etc..Only son and heir's grass (Oresitrophe rupifraga) is Saxifragaceae (Saxifragaceae) only son and heir's grass platymiscium (this belongs to for single), is mainly distributed on China Henan, Hebei, western Liaoning Province and Shanxi East, its habitat is very unique, is grown on the ground such as the mountain valley of 600 2000 meters of height above sea level, the dark and damp stone gap of steep cliff more, thus adopts Collection is very difficult；Among the people the effect of there are clearing away heat-damp and promoting diuresis, can be used for infantile enteritiss and have loose bowels with all herbal medicine, but its chemistry becomes Subsystem research and relevant biological activity are there is not yet any report.

Based on the present situation of prior art, present inventor prepares herb ethanol extract from this only son and heir's grass platymiscium, divides further From noval chemical compound therein, and for preparation treatment neurodegenerative diseases medicine.

Separate two noval chemical compounds obtaining：(7R, 8S, 8'S) -9-hydroxy-4,4'-dimethoxy-7,9'-epoxy-8,8'-lignan (changes Compound 1) and 3 β-hydroxy-15 β-O- β-D-glucopyranosyl-cholesta-5-ene (compound 2), through external activity screening Discovery is respectively provided with significant neuritis disease activity (IC₅₀It is respectively less than 10 μM).

Content of the invention

It is an object of the invention to provide for the defect of prior art, preparing herb ethanol extract from only son and heir's grass platymiscium, dividing further From noval chemical compound therein, and for preparation treatment neurodegenerative diseases medicine.

Specifically, the invention provides only son and heir's grass (Oresitrophe rupifraga Bunge) extract and preparation method thereof and Purposes in pharmacy, the especially purposes in preparation treatment neurodegenerative diseases medicine.

In the present invention, herb ethanol extract is prepared using only son and heir's grass platymiscium, separately noval chemical compound therein further, obtain chemical combination Thing 1 (lignanoid, 7R, 8S, 8'S) -9-hydroxy-4,4'-dimethoxy-7,9'-epoxy-8,8'-lignan) and compound 2 (cholesterol Glycosides, 3 β-hydroxy-15 β-O- β-D-glucopyranosyl-cholesta-5-ene), in embodiments of the invention, preferred acquisition Henan Rare plant only son and heir grass (Oresitrophe rupifraga Bunge) of the domestic Yuntai Shan Mountain in province Jiaozuo City Xiuwu County prepares herb alcohol Compound therein is separated, through external activity screening, result shows, described compound is respectively provided with significantly anti-nerve after extract Inflammatory activity (IC₅₀It is respectively less than 10 μM)；Can be used for the preparation treatment nervus retrogression disease such as Alzheimer and parkinson The medicine of disease.

Compound 1 and 2 of the present invention, its structural formula is as follows：

Only son and heir's grass extract active component of the present invention is obtained by following methods, first with only son and heir's grass herb as raw material, using molten Agent extraction method makes concentrated extract；In the solvent extraction method being adopted, can be single from water, methanol, ethanol, acetone etc. The mixing solvent of solvent or multi-solvents, reflux, extract, under Soakage extraction or heating condition at room temperature, extraction time is 2～3 times, Extracting solution is suspended in aqueous solution after concentrating, and first removes depigmentation with petroleum ether extraction, is extracted with ethyl acetate 2～3 times afterwards, warp Cross multiple column chromatography, monomeric compound 1 and compound 2 are obtained from the ethyl acetate layer component of only son and heir's grass.

The present invention is tested to the neuritis disease activity of prepared compound 1 and 2, and result shows, described compound 1 In the BV-2 microglia inducing with 2 pairs of lipopolysaccharide (LPS), the release of NO is respectively provided with significant inhibitory action (IC₅₀Value It is respectively 7.21 and 9.39 μM), and there is no toxic effect to cell survival rate, further, can be applicable to prevent and/or treat Multiple neurodegenerative diseases.

Compound 1 and 2 of the present invention can individually be applied or share, or is combined with suitable excipient, according to routine Method makes the treatment that oral or non-oral dosage forms is applied to neurodegenerative diseases.

The invention has the advantages that：

The chemical constitution novelty volume compound 1 and 2 being obtained from rare plant only son and heir grass；It is verified by experiments both to be respectively provided with significantly Neuritis disease activity, to neurodegenerative diseases, especially have a strong impact on life of elderly person quality Alzheimer and Parkinson has significant application prospect；Though both fractional doses from only son and heir's grass are less, according to its construction features, also Can by semi-synthetic (as with cholesterol as precursor) or complete synthesis be amplified producing and structure derivatization, application prospect is stronger.

Specific embodiment

With reference to specific embodiment, the present invention is further elaborated, but these embodiments have absolutely not any restriction to the present invention. Those skilled in the art will fall in claims to any variation made in present invention enforcement under the enlightenment of this specification In the range of.

Embodiment 1

Using only son and heir grass herb 3kg, pulverize, with 7L 90% methanol room temperature merceration 36 hours, totally 4 times, merge 4 extractions Liquid, concentrating under reduced pressure, obtain 383g extractum, the extractum obtaining is dissipated with the moisture of 1.2L, then use petroleum ether, acetic acid successively Ethyl ester and n-butanol extraction, by ethyl acetate portion 85.9g with big silicagel column (10 × 80cm；100-200mesh) carry out tentatively Segmentation, eluant is petroleum ether, ethyl acetate (from 5:1 to 0:1, v/v) and ethyl acetate, methanol are (from 20:1 to 0:1, V/v), 5 components Fr.1-Fr.5 are obtained；Fr.2 prepares HPLC [MeOH/H with half after adopting the multiple separation of silicagel column₂O 70:30,v/v；Flow velocity：3.0mL/min] obtain compound 1 (3.8mg, t_R=15.1min)；Fr.5 (6.7g) is through MCI post layer Analysis [MeOH/H₂O(3:7-1:0, v/v)] and repeatedly after normal phase silica gel column chromatography chromatography, by Sephadex LH-20 (CH₂Cl₂/MeOH,2:1) purification obtains compound 2 (2.4mg).

The spectrum of prepared compound and physicochemical data are as follows：

(7R, 8S, 8'S) -9-Hydroxy-4,4'-dimethoxy-7,9'-epoxy-8,8'-lignan (compound 1)：White powder；[α]D22+11.7(c 0.2, MeOH)；UV(MeOH)λmax(logε):226(4.23),276(3.36)nm；CD(c 0.91×10-3M,MeOH)λmax(Δε)216 (+0.61),231(–1.25)；IR(film)vmax:3414,2918,2849,1617,1512,1034cm-1；1H NMR(CDCl3,400MHz):δ 7.25 (2H, d, J=8.5Hz, H-2/H-6), 7.12 (2H, d, J=8.5Hz, H-2'/H-6'), 6.87 (2H, d, J=8.5Hz, H-3/H-5), 6.84 (2H, D, J=8.5Hz, H-3'/H-5'), 4.83 (1H, d, J=6.4Hz, H-7), 4.06 (1H, dd, J=8.0,7.2Hz, Ha-9'), 3.91 (1H, dd, J=7.6, 6.8Hz, Ha-9), 3.80 (3H, s, O-Me'), 3.79 (3H, s, O-Me), 3.76 (1H, overlapped, Hb-9), 3.73 (1H, dd, J=8.0,6.4Hz, Hb-9'), 2.91 (1H, dd, J=13.4,5.0Hz, H-7'), 2.73 (1H, m, H-8'), 2.58 (1H, dd, J=13.4,10.7Hz, H-7'), 2.39 (1H, m, H-8)；13C NMR(CDCl3,100MHz):δ159.0(C-4),159.8(C-4'),135.0(C-1),132.5(C-1'),129.6(C-2'),127.0 (C-2),114.0(C-3'),113.9(C-3),82.7(C-7),73.0(C-9'),61.0(C-9),55.3(O-Me),55.3(O-Me'),52.6(C-8),42.2 (C-8'),32.7(C-7').(+)ESIMS m/z 351.0[M+Na]+,679.2[2M+Na]+；HR-ESIMS m/z 351.1568[M+Na]+(calcd For C20H24NaO4,351.1567, △=0.5ppm).

3 β-hydroxy-15 β-O- β-d-glucopyranosyl-cholesta-5-ene (compound 2):White powder；[α]D22–30(c 0.1,CHCl3)； UV(CHCl3)λmax(logε):240(3.51),277(3.33)nm；IR(film)νmax:3387,2925,2868,1698,1259,1016,759 cm-1；1H and 13C NMR data is shown in Table 1；(+)ESIMS m/z 587.0[M+Na]+；HR-ESIMS m/z 587.3922[M+Na]+ (calcd for C33H56O7Na, 537.3918, △=0.7ppm).

Table 1.¹H(400MHz,J in Hz)and¹³C(100MHz)NMR Data of 2.

^aRecorded in C₅D₅N；^bRecorded in CDCl₃.

Embodiment 2：Neuritis disease active testing is tested

Mice little colloid BV-2 cell is purchased from American type culture collection (Manassas, VA, USA), and culture exists DMEM (Dulbecco's modified Eagle's medium) culture medium (includes 1800mg/L NaHCO₃, 10% (v/v) heat Hyclone (FBS), 100unit/mL penicillin G sodium salt and the 100 μ g/mL streptomycins of inactivation), 37 DEG C, 5%CO₂ Environment, in incubator；The yield of NO passes through Griess test kit (Beyotime Biotechnology, China) and measures, that is, BV-2 cell variable concentrations testing compound pretreatment 4h, is subsequently adding lipopolysaccharide (LPS, 1 μ g/ml, Sigma-Aldrich) Incubation 24h (blank group is not added with LPS).Draw culture supernatant, mix with the Griess reagent of equivalent, room temperature lucifuge standing 10 Minute, the OD value of each group is measured at 540nm wavelength with microplate reader (M200, TECAN, Austria GmbH, Austria), Use NaNO₂Draw standard curve, calculate the content of NO in sample；Data is carried out using one-way analysis of variance (ANOVA) Analysis is simultaneously represented with meansigma methodss ± SD, P<0.05, with L-NMMA as positive control, test result is as shown in table 2：

The inhibitory action of NO release in the BV-2 cell that the surveyed compound of table 2. induces to LPS

^aIC₅₀Value of concentration μM of of indicated compound that caused 50% of each compound was defined as the inhibition of NO production in LPS-stimulated BV-2 cells.

^bThe results are averages of three independent experiments,and the data were expressed as mean±SD.

^c50 μM of of each compound was expressed as a percentage % of untreated of Cell viability after treatment with control cells.

^dL-NMMA:positive control..

Claims

1. purposes in preparation treatment neurodegenerative diseases medicine for the compound 1 and 2 of formula,

2. the purposes as described in claim 1 is it is characterised in that described neurodegenerative diseases are Alzheimer (AD) Or parkinson (PD).

3. the purposes as described in claim 1 or 2 is it is characterised in that described compound 1 or 2 suppression neuroinflamation activity, IC₅₀It is respectively less than 10 μM.

4. the purposes as described in claim 1 is it is characterised in that described compound 1 and 2 is prepared by following methods,

With only son and heir's grass herb as raw material, concentrated extract is made using solvent extraction method；Wherein, from water, methanol, ethanol, Or the mixing solvent of acetone single solvent or described solvent, reflux, extract, under Soakage extraction or heating condition at room temperature, extract time Number is 2～3 times, and extracting solution is suspended in aqueous solution after concentrating, and is gone after depigmentation with petroleum ether extraction, it is extracted with ethyl acetate 2～ 3 times, through multiple column chromatography, monomeric compound 1 and compound 2 are obtained from the ethyl acetate layer component of only son and heir's grass.

5. the purposes as described in claim 1 is it is characterised in that described only son and heir's grass is the domestic cloud in Henan Province Jiaozuo City Xiuwu County Plant only son and heir grass (Oresitrophe rupifraga Bunge) in the Taishan.

6. the purposes as described in claim 1 and 2 it is characterised in that described compound 1 and 2 individually apply or Share.

7. the purposes as described in claim 1 it is characterised in that compound 1 and 2 be combined with excipient respectively make tablet, Capsule, granule or injection.