CN101791336B - Extract from long pepper and preparation method and application thereof - Google Patents
Extract from long pepper and preparation method and application thereof Download PDFInfo
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- CN101791336B CN101791336B CN2010101444711A CN201010144471A CN101791336B CN 101791336 B CN101791336 B CN 101791336B CN 2010101444711 A CN2010101444711 A CN 2010101444711A CN 201010144471 A CN201010144471 A CN 201010144471A CN 101791336 B CN101791336 B CN 101791336B
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Abstract
The invention discloses an extract from long pepper and a preparation method and application thereof. The preparation method provided by the invention comprises the following steps: 1) extracting dry long pepper fruits by using 70 to 95 percent ethanol solution; 2) concentrating the extracted solution obtained in the step 1) to form concentrated solution with the relative density of 1 to 1.1; 3) diluting the concentrated solution obtained in the step 2) by using water, centrifuging the diluted solution, and adsorbing the centrifuged supernatant by using a D101 macroporous absorption resin column; and 4) eluting the D101 macroporous absorption resin column which is obtained in the step 3) and adsorbs the supernatant by using 20 percent ethanol, then eluting the D101 macroporous absorption resin column by using 70 percent ethanol of in a volume which is 20 times that of a column volume, and collecting the elution from the third to twentieth column volume so as to obtain the protected extract from the long pepper. In SH-SY5Y cells, SH-SK-N cells and MN9D cells, the extract of the invention has remarkable difference on nerve protection effect of oxidative damage caused by rotenone resistance compared with a rotenone model set, and has better medicinal effect than a positive medicinal set.
Description
Technical field
The present invention relates to the field of Chinese medicines, particularly from the extract of Fructus Piperis Longi and preparation and application.
Background technology
Parkinson disease (Parkinson ' s Disease, PD) be one of modal nervous system degenerative disease clinically.Along with the aging of China's population, the sickness rate of PD is in rising trend.The healthy net report of USA I S, the global expenditure that is used for PD had 2,500,000,000 U.S. dollars in 2004, and China has 3,000,000 PD patients at present.PD morbidity back disability rate height has become the great social problem that influences China's aging population health level and quality of life.Modern medicine is treated Parkinsonian means and mainly contained: Drug therapy, operation method, cell transplantation, transgenic technology etc., because many effects limit, Drug therapy is still main method at present.The medicine of the control PD clinical symptoms the best of generally acknowledging is a levodopa class medicine at present,, this medicine can not control the process of PD but can only improving symptom, along with this medicine is used the prolongation of time and the increasing of dosage, its therapeutical effect is more and more littler, and toxicity is increasing.In recent years, people invest traditional Chinese medicine to sight gradually, in the hope of therefrom seeking out multipath, the Parkinsonian new drug of many target treatments and new method.Studies confirm that in a large number Chinese medicine reaches the Parkinsonian purpose of treatment by effects such as protection nigral cell, raising neurotransmitter content, inhibited oxidation stress, hypo irritability toxicity; and some Chinese medicine is to reducing and prevention is taken Western medicine side effect such as levodopa and also brought into play certain curative effect, even begun deeply to inquire into the Chinese medicine mechanism of action from the molecular biology angle.These illustrate that all the Chinese medicine parkinson disease not only are confined to traditional clinical observation, but more and more pay attention to experimentation, go to study its curative effect from parkinson disease pathogenesis, Pathophysiology and genetics angle.
The flavour of a drug suffering of Fructus Piperis Longi medicine, hardship, warm in nature.Return liver, spleen channel.Invigorate blood circulation promoting the circulation of QI, pain relieving.Show the chemical compound that contains abundant alkaloid, amide-type, lignanoids, terpenoid, sterols and other class in this medicine by The Chemical Constituents, wherein alkaloid and amide-type are about 35 kinds, and especially the content of piperine is no less than 2.5%.The bibliographical information piperine has many-sided biological activitys such as calmness, blood pressure lowering, convulsion, antiinflammatory, antioxidation and antidepressant, and may become the brain metabolism improving medicine for the treatment of multiple cognitive disorder, and can obviously increase the content of monoamine neurotransmitter 5-HT in the mouse brain.Bibliographical information the calmness of this medicine, convulsion, hypnosis, hypotensive effect, illustrate that it has the effect similar with sedative, this class medicine all has the Antiparkinsonian effect, main pharmacological is antagonism or improves parkinson sample sign.
Yet there are no total alkaloids and the correlational study of piperine aspect the parkinson disease treatment in the report Fructus Piperis Longi in the document.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of extract from Fructus Piperis Longi.
Preparation method provided by the invention comprises the steps:
1) getting the Fructus Piperis Longi dry fruit, is that 8-10 70-95% (the being percent by volume) ethanol water doubly of described Fructus Piperis Longi dry fruit extracts described Fructus Piperis Longi dry fruit as extractant with weight, obtains extracting solution;
2) extracting solution that step 1) is obtained is condensed into the concentrated solution that relative density is 1-1.1;
3) with step 2) the concentrated solution dilute with water that obtains, centrifugal, with the supernatant after centrifugal through the absorption of D101 macroporous adsorptive resins;
4) absorption that step 3) is obtained the D101 macroporous adsorptive resins of described supernatant with 10-20% ethanol water (percent by volume) eluting of 5-15 times of column volume, with the D101 macroporous adsorptive resins that strips 60-70% ethanol water (percent by volume) eluting with 10-20 times of column volume, collect the eluent of the 3rd to 20 column volume, promptly obtain extract (called after extract E) from Fructus Piperis Longi.
In step 1), in order to extract fully, the Fructus Piperis Longi dry fruit can be pulverized earlier, extracts again, and the number of times of said extracted can be 3 times that described extractant is the ethanol water of 85% (percent by volume).
In step 2) in, described concentrate is at 50-70 ℃ of following concentrating under reduced pressure, preferably concentrates under 60 ℃, vacuum 0.01Pa; The relative density of described concentrated solution is 1.08.
In step 3), described dilution is meant step 2) the concentrated solution thin up, concentrated solution adds gross weight behind the water and the ratio of described Fructus Piperis Longi dry fruit quality is 1: 1; Described centrifugal be under the 1000g-2000g, centrifugal 10 minutes.
In step 4), the 10-20% ethanol water eluting of described 5-15 times column volume is 20% ethanol water eluting of 10 times of column volumes preferably; In the step 5), the 60-70% ethanol water eluting of 10-20 times of column volume is 70% ethanol water eluting of 20 times of column volumes preferably.
After described step 5) obtains the eluent of the 3rd to 20 column volume, can carry out drying, exsiccant step is as follows: with the eluent concentrating under reduced pressure of collecting, vacuum lyophilization then.
The extract from Fructus Piperis Longi that arbitrary above-mentioned method prepares (extract E) also belongs within protection scope of the present invention.
The application of above-mentioned extract E in the medicine of preparation prevention or treatment Parkinson disease also belongs within protection scope of the present invention.
The application of above-mentioned extract E in the product of the cell survival rate of the external parkinson disease cell model of preparation raising also belongs within protection scope of the present invention.
The rotenone that above-mentioned parkinson disease cell model is 100nM is to the cell model of SH-SY5Y, SH-SK-N or the formation of MN9D cell line.
The application of above-mentioned extract E in the cell survival rate that improves external parkinson disease cell model also belongs within protection scope of the present invention.
The rotenone that above-mentioned parkinson disease cell model is 100nM is to the cell model of SH-SY5Y, SH-SK-N or the formation of MN9D cell line; The concentration of described extract E is 12.5-2.0g crude drug Fructus Piperis Longi/ml, and preferably 12.5-50mg crude drug Fructus Piperis Longi/ml is more preferably 25mg crude drug Fructus Piperis Longi/ml or 50mg crude drug Fructus Piperis Longi/ml.
Experimental results show that: add the administering mode protection best results that rotenone (final concentration 100nM) is cultivated 24h altogether again after adding drug treating 24h earlier; Fructus Piperis Longi ethanol extract, Fructus Piperis Longi water extract all have the certain protection effect to the SH-SY5Y cell (the inductive external PD model of oxidative damage) of rotenone damage; wherein 50mg/ml, 25mg/ml, the isocyatic protectiveness effect of 12.5mg/ml are better; the protectiveness effect of the concentration of piperine monomer 5ug/ml, 2.5ug/ml, 1.25ug/ml is better, and wherein the protective effect of Fructus Piperis Longi ethanol extract and Fructus Piperis Longi water extract is better.
Be further to determine the neuroprotective of Fructus Piperis Longi ethanol extract effective site, adopt MTT to detect cell viability again, the effectiveness and the concentration thereof at the various eluting position of Fructus Piperis Longi ethanol extract have been carried out primary dcreening operation.In SH-SY5Y cell, SH-SK-N cell and MN9D cell, the neuroprotective drug effect and the rotenone model group of the oxidative damage that extract E antagonism rotenone causes have significant difference, and drug effect is better than the positive drug group.
Description of drawings
Fig. 1 is time window and concentration window (100nM concentration is handled 24 hours observation of cell vigor), the wherein A of medicine antagonism rotenone damage SH-SY5Y cell, B, C is respectively the Fructus Piperis Longi ethanol extract, the monomeric different experiments group of the piperine that extracts in Fructus Piperis Longi water extract and the Fructus Piperis Longi.
Fig. 2 further detects the neuroprotective drug effect for the drug extract that will just sift out in SH-SY5Y cell line; be illustrated as add extract 24H earlier after; add 24 hours MTT of 100nM rotenone processing and send out detection cell viability (Fig. 2 A); LDH (lactic acid dehydrogenase) detection kit detects LDH release rate (Fig. 2 B); * representative and rotenone group are than p＜0.01, and * represents p＜0.05.
Fig. 3 further detects the cellular morphology of neuroprotective drug effect for the drug extract that will just sift out in SH-SY5Y cell line.
Fig. 4 further detects the neuroprotective drug effect for the drug extract that will just sift out in SH-SK-N cell line; be illustrated as add extract 24H earlier after; add 24 hours MTT of 100nM rotenone processing and send out detection cell viability (Fig. 4 A); LDH (lactic acid dehydrogenase) detection kit detects LDH release rate (Fig. 4 B); * representative and rotenone group are than p＜0.01, and * represents p＜0.05.
Fig. 5 further detects the cellular morphology of neuroprotective drug effect for the drug extract that will just sift out in SH-SK-N cell line.
Fig. 6 further detects the neuroprotective drug effect for the drug extract that will just sift out in MN9D cell line; be illustrated as add extract 12H earlier after; add 12 hours MTT of 100nM rotenone processing and send out detection cell viability (Fig. 6 A); LDH (lactic acid dehydrogenase) detection kit detects LDH release rate (Fig. 6 B); * representative and rotenone group are than p＜0.01, and * represents p＜0.05.
Fig. 7 adds the result that the 100nM rotenone is handled 12 hours cellular morphology for add extract 12H earlier in MN9D cell line after.
Fig. 8 is the collection of illustrative plates of the HPLC of extract E.
The specific embodiment
The invention will be further described below in conjunction with specific embodiment, but the present invention is not limited to following examples.
Among the following embodiment, if no special instructions, be conventional method.
Cell line used among the present invention is as follows:
SH-SY5Y is the neuroblastoma cell system in people source, available from the article No. CRL-2266 of ATCC company (American Type CultureCollection)
TM
SH-SK-N is the neuroblastoma cell system in people source, available from the article No. HTB-11 of ATCC company (American Type CultureCollection)
TM
MN9D is that the former generation embryo veutro midbrain cell and the nervelet blastoma cell line N18TG2 in mice source merges the hybridoma cell line that forms, available from Novartis company.
Test method of the present invention is as follows:
Detect the method (MTT) of cell viability: be a kind of method that detects cell survival and growth.It detects principle is that succinate dehydrogenase in the living cells mitochondrion can make exogenous MTT be reduced to water-insoluble bluish violet crystallization first a ceremonial jade-ladle, used in libation (Formazan) and is deposited in the cell, and dead cell does not have this function.Reacting cells vigor (this tests all MTT available from Sigma company) whereby.Concrete steps:
1) collects the logarithmic (log) phase cell, adjust concentration of cell suspension, divide, every hole 100 μ l, cell density 10000-15000/hole in 96 orifice plates.
2) put 37 ℃, 5%CO2 incubator and cultivate and make cell attachment, cultivated 6-24 hour.
3) adding waits to sieve sample and treatment conditions.
4) dispose after, the careful suction removed supernatant, adds 10ulMTT solution (5mg/ml, i.e. 0.5%MTT), continues 37 ℃ and continues cultivation 4h.
6) sop up supernatant then, every hole adds the 100ul dimethyl sulfoxide, puts low-speed oscillation 10min on the shaking table, and crystal is fully dissolved.Measure the light absorption value in each hole at enzyme-linked immunosorbent assay instrument 490nm place.
LDH (lactic acid dehydrogenase) release rate is the index of reacting cells survival: LDH (lactic acid dehydrogenase) is a kind of stable protein, is present in the Normocellular kytoplasm, in case damaged membrane, LDH promptly is released to the extracellular; LDH catalysis lactic acid forms pyruvate, and the crystalline material of INT (tetrazolium salt) reaction formation purple, can detect by the 490nm microplate reader.By detecting the activity of LDH in the cells and supernatant, can judge that the impaired degree of cell originally tests used LDH detection kit available from Roche Holding Ag's article No.: Cat.No.11 644,793 001.) concrete steps see description.
One, preparation
The Fructus Piperis Longi dry fruit is a crude drug in the present embodiment, can be available from pharmacy.
The preparation of Fructus Piperis Longi water extract: get the Fructus Piperis Longi dry fruit, be ground into coarse powder, with the purified water extraction of 8～10 times of medical material weight three times, each 1 hour, filter, merging filtrate, concentrating under reduced pressure is made the Fructus Piperis Longi water extract of 2g crude drug/ml.
The preparation of Fructus Piperis Longi ethanol extract: get the Fructus Piperis Longi dry fruit, be ground into coarse powder, extract three times with 95% alcoholic solution of 8～10 times of medical material weight, each 1 hour, filter merging filtrate, to there not being alcohol, make the Fructus Piperis Longi alcohol extract of 2g crude drug/ml in 60 ℃ of following decompression recycling ethanols with purified water.
The monomeric preparation of piperine in the Fructus Piperis Longi: content is greater than 90% piperine monomer.Get the Fructus Piperis Longi dry fruit, be ground into coarse powder, with 95% ethanol extraction of 8～10 times of medical material weight three times, each 1 hour, filter, merging filtrate, in 60 ℃ of following decompression recycling ethanols to there not being pure extractum, extractum is dissolved in an amount of water dissolving fully after, use petroleum ether successively, dichloromethane, ethyl acetate, n-butanol extraction, solvent is reclaimed at the dichloromethane extraction position get extractum, disperse with the methanol heating for dissolving, cooled insoluble matter dissolves with dichloromethane, solution behind the elimination insoluble matter is placed, separate out crystal, carry out recrystallization again, obtain content greater than 90% piperine.
Two, screening active ingredients
1,3 kinds of medicines that obtain at step 1 carry out the screening of best administration time and activity respectively:
Fructus Piperis Longi ethanol extract, Fructus Piperis Longi water extract reach determining with the time window of piperine monomer pair cell oxidative damage protective effect and concentration window, the cell strain that adopts is SH-SY5Y, the cell culture fluid that adopts is that (the DMEM/F12 culture medium can be available from Gibco company, article No. 12400-024 for the DMEM/F12+10% hyclone; Other 10% hyclone is meant that final concentration is 10%, is percentage by volume) (cell culture fluids of using are all identical with the culture fluid here in all experiments).Rotenone damage contrast: the cell culture fluid that contains the 100nM rotenone.
Fructus Piperis Longi ethanol extract final concentration in 96 orifice plates is respectively 50mg/ml, 25mg/ml, 12.5mg/ml and 6.25mg/ml; Fructus Piperis Longi water extract final concentration in 96 orifice plates is respectively 50mg/ml, 25mg/ml, 12.5mg/ml and 6.25mg/ml; Piperine monomer final concentration in 96 orifice plates is respectively 20ug/ml, 10ug/ml, 5ug/ml, 2.5ug/ml, 1.25ug/ml and 0.625ug/ml.
Three kinds of medicines carry out following three experiments (composition of every kind of extract does not inform that experimental implementation person only gives the medicine numbering, to avoid the interference of artificial subjective factor) respectively:
A. add the various extracts of Fructus Piperis Longi earlier and add rotenone again
A, the 96 orifice plate cells that inoculation is good are removed culture fluid, add the medicine of above-mentioned respective concentration, hatch 24 hours.
B, remove medicine, add rotenone (final concentration is 100nM in 96 orifice plates), hatched 24 hours.Normal cell contrast: normal SH-SY5Y cell.Carrying out mtt assay then detects.
B. add dosing behind the rotenone earlier
The 96-well plate cell that inoculation is good is removed culture fluid, adds rotenone solution (final concentration is 100nM in 96 orifice plates), hatches 24 hours.Remove rotenone solution, add the relative medicine extract solution, hatched the normal cell matched group 24 hours: normal SH-SY5Y cell.Carrying out mtt assay then detects.
C. dosing simultaneously and rotenone
The 96 orifice plate cells that inoculation is good are removed culture fluid, add relative medicine and rotenone solution (rotenone final concentration in 96 orifice plates is 100nM) and hatch 24 hours.Normal cell contrast: normal SH-SY5Y cell.Rotenone contrast: 100nM rotenone contrast.Carrying out mtt assay then detects.
2, experimental result
The result adds the administering mode protection best results that rotenone (final concentration 100nM) is cultivated 24h altogether earlier as shown in Figure 1 again behind the adding drug treating 24h; Fructus Piperis Longi ethanol extract, Fructus Piperis Longi water extract all have the certain protection effect to the SH-SY5Y cell (the inductive external PD model of oxidative damage) of rotenone damage; wherein 50mg/ml, 25mg/ml, the isocyatic protectiveness effect of 12.5mg/ml are better; the protectiveness effect of the concentration of piperine monomer 5ug/ml, 2.5ug/ml, 1.25ug/ml is better, and wherein the protective effect of Fructus Piperis Longi ethanol extract and Fructus Piperis Longi water extract is better.
Be further to determine the neuroprotective of Fructus Piperis Longi ethanol extract effective site, adopt MTT to detect cell viability again, the effectiveness and the concentration thereof at the various eluting position of Fructus Piperis Longi ethanol extract have been carried out primary dcreening operation.
One, the preparation at various eluting position
Fructus Piperis Longi extract A: get the Fructus Piperis Longi dry fruit, be ground into coarse powder, extract three times with 95% alcoholic solution of 8～10 times of medical material weight, each 1 hour, filter, merging filtrate to there not being alcohol, is made the Fructus Piperis Longi alcohol extract of 2g crude drug/ml in 60 ℃ of following decompression recycling ethanols.
Fructus Piperis Longi extract B: get the Fructus Piperis Longi dry fruit, be ground into coarse powder, with the purified water extraction of 8～10 times of medical material weight three times, each 1 hour, filter, merging filtrate, concentrating under reduced pressure is made the Fructus Piperis Longi water extract of 2g crude drug/ml.
Piperine C (extract C): content is greater than 90% piperine.Get the Fructus Piperis Longi dry fruit, be ground into coarse powder, with 95% ethanol extraction of 8～10 times of medical material weight three times, each 1 hour, filter, merging filtrate, in 60 ℃ of following decompression recycling ethanols to there not being pure extractum, after extractum is dissolved in an amount of water dissolving fully, use petroleum ether successively, dichloromethane, ethyl acetate, n-butanol extraction reclaims solvent to the dichloromethane extraction position and gets extractum, disperses with the methanol heating for dissolving, cooled insoluble matter, with the dichloromethane dissolving, the solution behind the elimination insoluble matter is placed, and separates out crystal, carry out recrystallization again, obtain content greater than 90% piperine.
Total alkaloids---each eluting position (D, E, J): get the Fructus Piperis Longi dry fruit, be ground into coarse powder, 85% (percent by volume) alcoholic solution with 8～10 times of medical material weight extracts three times, each 1 hour, filters, merging filtrate, decompression (vacuum 0.01Pa) recovery ethanol to relative densities are 1.08 concentrated solution under 60 ℃, the concentrated solution dilute with water, and the mass ratio of quality after the dilution and Fructus Piperis Longi dry fruit is 1: 1, then under 1500g, centrifugal 10 minutes.Supernatant after centrifugal through macroporous adsorptive resins (D101 type) absorption, with 20% (percent by volume) ethanol elution of 10 times of column volumes, is obtained eluting position D earlier; Use 70% ethanol elution of 20 times of column volumes then, collect the eluent of the 3rd to 20 column volume, obtaining the eluting position is E; Use 85% (percent by volume) ethanol elution at last, collect eluent and obtain eluting position J.With each eluting position concentrating under reduced pressure, vacuum lyophilization, the different eluting position (obtaining extract D, E and J is pressed powder or mastic, before doing the cell screening, is mixed with the solution of 2g crude drug/ml with aforesaid cell culture fluid) that promptly gets the Fructus Piperis Longi total alkaloids.
Wherein the collection of illustrative plates of the HPLC of extract E as shown in Figure 8, HPLC chromatographic condition: chromatographic column: Agilent EclipseXDB-C
18(5 μ m, 4.6mm * 250mm); Mobile phase: 0.25% formic acid-methanol (35: 65); Flow velocity: 1.0mLmin-1; Detect wavelength: 343nm; Sample size: 20 μ L; Column temperature: 40 ℃.
Each above-mentioned extract (A, B, D, E and J) is diluted to A1, B1, D1, E1 and the J1 of 50mg crude drug/ml, on the basis of this concentration, carry out 2 times ratio of two term then and dilute A2, B2, D2, E2 and the J2 that obtains 25mg crude drug/ml, also obtain A3, B3, D3, E3 and the J3 of 12.5mg crude drug/ml; Extract C is diluted to the C1 of 5ug crude drug/ml, the C3 of the C2 of 2.5ug crude drug/ml and 1.25ug crude drug/ml.
Two, active Preliminary screening
1, experimental procedure:
With the various extracts of Fructus Piperis Longi and each component after the dilution, the best administration time point (i.e. the 96 orifice plate cells that inoculation is good are removed culture fluid, add Fructus Piperis Longi extract effect 24h earlier, add rotenone again and handle 24h) so that primary dcreening operation is determined detects cell viability.
Wherein not add extract, only adding 24 hours group of 100nM rotenone processing is the rotenone model group;
After adding tocopherol 24h, add the positive medicine group of group that the 100nm rotenone is handled 24h.
2, the experimental result result is as shown in table 1, and the neuroprotective drug effect and the rotenone model group of the oxidative damage that the extract antagonism rotenone of 3 concentration of E component causes have significant difference, and drug effect is better than the positive drug group.
Table 1. is in the SH-SY5Y cell, and cell viability detects
Annotate: △ △ representative and rotenone group are than p＜0.01, and drug effect is better than the positive drug group.
Three, in 3 kinds of cell lines, B, C, D, E and the active of J group are further screened
1, the screening active ingredients in the SH-SY5Y cell
Component after B, C, D and four components of E and each dilution is added respectively in the SH-SY5Y cell line behind the 24h, add the 100nM rotenone again and handle 24h, detect vigor and detect the LDH release rate with mtt assay.
3 matched group: control groups wherein are set are the normal cell matched group, the Rotenone group is handled 24 hours rotenone MODEL DAMAGE group for only having added the 100nM rotenone, the Tocopherol group is that 80 μ g/ml tocopherols (positive drug control group) are handled 24h for adding working concentration earlier, add rotenone again and handle 24h, detect cell viability.
The result as shown in Figure 2, the survival rate and the rotenone model group of the SH-SY5Y cell under the E component of 2 concentration (E1 and E2) is handled have significant difference (Fig. 2 A), and the LDH release rate (Fig. 2 B) of the SH-SY5Y cell under the E component of 2 concentration (E1 and the E2) processing also has significant difference with the rotenone model group.
The photo of cellular morphology as shown in Figure 3, A:SY5Y Cell (A represents normal SH-SY5Y cell) wherein, B:Tocopherol (the B representative adds in the SH-SY5Y cell and adds rotenone 100nM processing 24h again after Tocopherol handles 24h), C:Extract E (C representative adding E group extract optium concentration in the SH-SY5Y cell is the cell that adds rotenone 100nM processing again after 50mg/ml handles 24h), D:Rotenone, 100nM (D has only added the rotenone of 100nM and handled 24 hours cell).
This shows that the neuroprotective drug effect and the rotenone model group of the oxidative damage that E component antagonism rotenone causes have significant difference.
2, the screening active ingredients in the SH-SK-N cell
Component after B, C, E and four components of J and each dilution is added respectively in the SH-SK-N cell line behind the 24h, add the 100nM rotenone again and handle 24h, detect vigor and detect the LDH release rate with mtt assay.
Wherein mtt assay detection vigor is provided with 3 matched group: control group and is the normal cell matched group; The Rotenone group is handled 24 hours rotenone MODEL DAMAGE group for only having added the 100nM rotenone; The Tocopherol group is that 80 μ g/ml tocopherols (positive drug control group) are handled 24h for adding people's working concentration earlier, adds rotenone again and handles 24h, detects cell viability.
And the test experience of LDH release rate has also been established another kind of positive drug CoQ10: a kind of in fact antioxidative mitochondrion protecting agent of generally acknowledging is the ingredient of mitochondrial respiratory chain.2 concentration 20uM and 100uM have been adopted.
The result as shown in Figure 4, the survival rate and the rotenone model group of the SH-SKN cell under the E component of 2 concentration (E1 and E2) is handled have significant difference (Fig. 4 A), and the LDH release rate of the SH-SKN cell under B, C, E and J group are handled all also has significant difference (Fig. 4 B) with the rotenone model group.
The photo of cellular morphology as shown in Figure 5, A:SH-SKN Cell (A represents normal SH-SKN cell) wherein, B:Tocopherol (the B representative adds in the SH-SY5Y cell and adds rotenone 100nM processing 24h again after Tocopherol handles 24h), C:Extract E (C representative adding E group extract optium concentration in the SH-SKN cell is the cell that adds rotenone 100nM processing again after 50mg/ml handles 24h), D:Rotenone, 100nM (D has only added the rotenone of 100nM and handled 24 hours cell).
This shows that the neuroprotective drug effect and the rotenone model group of the oxidative damage that E component antagonism rotenone causes have significant difference.
3, the screening active ingredients in the MN9D cell
B, C, E and four components of J (B, E, J are the identical 50mg/ml of concentration, and C is 5ug/ml) are added respectively in the MN9D cell line behind the 12h, add the 100nM rotenone again and handle 12h, detect vigor and detect the LDH release rate with mtt assay.
4 matched group: control groups wherein are set are the normal cell matched group; The Rotenone group is handled 24 hours rotenone MODEL DAMAGE group for only having added the 100nM rotenone; The Tocopherol group is that 80 μ g/ml tocopherols (positive drug control group) are handled 24h for adding people's working concentration earlier, adds rotenone again and handles 24h, detects cell viability; The CoQ10 group adds rotenone processing mtt assay detection in 12 hours cell viability again for adding people CoQ1020uM processing 12h earlier.
The result as shown in Figure 6A, the survival rate of the MN9D cell after B, C, E handle is significantly higher than that there were significant differences with rotenone damage group.
And the test experience of LDH release rate is provided with positive drug CoQ10 and tocopherol.
The result is shown in Fig. 6 B, and the survival rate and the rotenone model group of the MN9D cell under the E component is handled have significant difference (Fig. 6 B), and the LDH release rate of the SH-SY5Y cell under B, E group is handled all also has significant difference with the rotenone model group.
The photo of cellular morphology as shown in Figure 7, A:MN9D Cell (A represents normal MN9D cell) wherein, B:Tocopherol (the B representative adds in the MN9D cell and adds rotenone 100nM processing 12h again after Tocopherol handles 12h), C:Extract E (the C representative adds the cell that adds rotenone 100nM processing again behind the E group extract-treated 12h in the MN9D cell), D:Rotenone, 100nM (D has only added the rotenone of 100nM and handled 24 hours cell).
This shows that the neuroprotective drug effect and the rotenone model group of the oxidative damage that E component antagonism rotenone causes have significant difference.
Claims (12)
1. the preparation method of extract from Fructus Piperis Longi comprises the steps:
1) getting the Fructus Piperis Longi dry fruit, is that the 8-10 percent by volume doubly of described Fructus Piperis Longi dry fruit is that the ethanol water of 70-95% extracts described Fructus Piperis Longi dry fruit as extractant with weight, obtains extracting solution;
2) extracting solution that step 1) is obtained is condensed into the concentrated solution that relative density is 1-1.1;
3) with step 2) the concentrated solution dilute with water that obtains, centrifugal, with the supernatant after centrifugal through the absorption of D101 macroporous adsorptive resins;
4) absorption that step 3) is obtained the D101 macroporous adsorptive resins of described supernatant be the ethanol water eluting of 10-20% with the percent by volume of 5-15 times of column volume, is the ethanol water eluting of 60-70% with the D101 macroporous adsorptive resins that strips with the percent by volume of 10-20 times of column volume, collect the eluent of the 3rd to 20 column volume, promptly obtain extract from Fructus Piperis Longi.
2. method according to claim 1 is characterized in that:
In the step 1), the number of times of described extraction is 3 times, and described extractant is that percent by volume is 85% ethanol water;
Step 2) in, described concentrating is at 50-70 ℃ of following concentrating under reduced pressure; The relative density of described concentrated solution is 1.08.
3. method according to claim 2 is characterized in that:
Step 2) in, described concentrate is to concentrate under 60 ℃, vacuum 0.01Pa.
4. according to arbitrary described method among the claim 1-3, it is characterized in that: in the step 3), described dilution is meant step 2) the concentrated solution thin up, concentrated solution adds gross weight behind the water and the ratio of described Fructus Piperis Longi dry fruit quality is 1: 1; Described centrifugal be under 1000g-2000g, centrifugal 10 minutes.
5. method according to claim 1 is characterized in that: in the step 4), the absorption that step 3) is obtained the D101 macroporous adsorptive resins of described supernatant be 20% ethanol water eluting with the percent by volume of 10 times of column volumes; 70% ethanol water eluting with the D101 macroporous adsorptive resins that strips with the percent by volume of 20 times of column volumes.
6. the extract that arbitrary described method prepares among the claim 1-5 from Fructus Piperis Longi.
7. the application of the described extract of claim 6 in the medicine of preparation prevention or treatment Parkinson disease.
8. the application of the described extract of claim 6 in the product of the cell survival rate of the external parkinson disease cell model of preparation raising.
9. application according to claim 8 is characterized in that: the rotenone that described parkinson disease cell model is 100nM is to the cell model of SH-SY5Y, SH-SK-N or the formation of MN9D cell line.
10. the application of the described extract of claim 6 in the cell survival rate that improves external parkinson disease cell model.
11. application according to claim 10 is characterized in that: the rotenone that described parkinson disease cell model is 100nM is to the cell model of SH-SY5Y, SH-SK-N or the formation of MN9D cell line; The concentration of the described extract of described claim 6 is 12.5-50mg crude drug Fructus Piperis Longi/ml.
12. application according to claim 11 is characterized in that: the rotenone that described parkinson disease cell model is 100nM is to the cell model of SH-SY5Y, SH-SK-N or the formation of MN9D cell line; The concentration of the described extract of described claim 6 is 25mg crude drug Fructus Piperis Longi/ml or 50mg crude drug Fructus Piperis Longi/ml.
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| CN102908401A (en) * | 2012-09-21 | 2013-02-06 | 首都医科大学 | Application of fructus piperis longi on preparation of medicine for inhibiting neuroinflammation |
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| JP5889546B2 (en) * | 2011-04-28 | 2016-03-22 | 丸善製薬株式会社 | Hexosaminidase release inhibitor, stem cell growth factor mRNA expression increase inhibitor, hydrogen peroxide cell disorder preventive / ameliorating agent and glutathione production promoter |
| CN106389410A (en) * | 2016-12-01 | 2017-02-15 | 首都医科大学 | Application of piperlonguminine in preparation of drugs for treating Parkinson's disease |
| CN107827864B (en) * | 2017-11-30 | 2020-02-07 | 宁夏医科大学 | Method for separating chemical components of methylene chloride extraction part of piper longum composition and application |
| CN110013504A (en) * | 2019-05-13 | 2019-07-16 | 首都医科大学 | Fructus piperis longi total alkaloid is adjusting intestinal flora and is improving the application in Parkinson's disease and Patients with Depression |
| CN112675221A (en) * | 2021-01-05 | 2021-04-20 | 首都医科大学 | Application of long pepper total alkaloids and piperine in preparation of adjuvant therapy medicines for Parkinson's disease |
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| CN101554409B (en) * | 2008-04-10 | 2011-11-09 | 周亚伟 | Long pepper alkaloid and preparation method, preparation and application thereof |
| CN101352487B (en) * | 2008-08-04 | 2011-04-06 | 北京宝泰宁堂医药科技有限公司 | Long pepper extract, preparation method thereof, medicament composition and uses thereof |
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| CN102908401B (en) * | 2012-09-21 | 2016-04-13 | 首都医科大学 | Fructus Piperis Longi suppresses the application in the medicine of neuroinflamation in preparation |
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