CN101979021B - Compound extractive with antioxidation activity - Google Patents
Compound extractive with antioxidation activity Download PDFInfo
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- CN101979021B CN101979021B CN2010105333065A CN201010533306A CN101979021B CN 101979021 B CN101979021 B CN 101979021B CN 2010105333065 A CN2010105333065 A CN 2010105333065A CN 201010533306 A CN201010533306 A CN 201010533306A CN 101979021 B CN101979021 B CN 101979021B
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Abstract
The invention discloses a compound extractive obtained from a Baimai powder compound by using a quick preparation technology and antioxidation activity thereof. The extractive has strong antioxidation activity, especially has good scavenging effect for free radicals, wherein the ability of scavenging the free radicals of part of components is far higher than the free radial scavenging agent Edaravone. The excess production of the free radials is an important reason for the generation and the development of multiple diseases, so the extractive has a potential application value.
Description
Technical field
The present invention relates to one group of compound extract, particularly relate to the antioxidant activity of this group extract.
Background technology
Free radical is the metabolite of body, discovers that the incidence and development of a lot of diseases is all excessive relevant with the body free radical.The generation of free radical can cause lipid, protein and nucleic acid peroxidating, makes membrane structure impaired.After apoplexy took place, especially behind the cerebral ischemia re-pouring, free radical produced in a large number, and neuron is caused major injury, was that cerebral edema forms and apoptotic major reason.Antioxidant prevents lipid peroxidation through removing excessive free radical, can play a protective role to cranial nerve cell.
The ethnic groups medicine is the important component part of China's conventional medicament; Its aboundresources, characteristic is distinct, and is theoretical original; Condensed thousands of year over ethnic groups working people and the Nature fight, the countless invaluable experiences of resisting with disease; But, still be in state original, that fall behind, demand urgently excavating, protecting not by external world's understanding.Therefrom find to have the strong anti-oxidation Natural antioxidant active and high security and be used for the apoplexy treatment, should be significant.
The lung-pulse looses and is the mongolian medicine compound recipe, is made up of 19 flavor medical materials such as Fructus Chebulae, Rhizoma Picrorhizae, Artemisia frigida Willd..This side regulates and conspicuously hands over and strive according to, sticking, heat, solid three, urgees balance; Have blood circulation promoting and blood stasis dispelling, tranquillizing and allaying excitement, correction stupor, control irritability, mediation the lung-pulse, Hei Mai; The effect of the continuous life of refreshment can be controlled brain and suffer from, more the sea of the lung-pulse; Being used for the treatment of apoplexy clinically, is to have the national medicine that exploitation is worth.
Summary of the invention
The object of the present invention is to provide one group of the lung-pulse compound extract that looses.
For describing the characteristic of this group extract, the invention provides preparation method of extract, but method for preparing itself is not protected.
The extract that the objective of the invention is to be provided has antioxidant activity, is with a wide range of applications, and especially in the treatment of apoplexy, possibly have potential clinical value.
Preparation method of extract of the present invention:
Take by weighing medical material (by weight) by the diffusing compound recipe proportioning of the lung-pulse: the Fructus Chebulae is 30～60 weight portions; Rhizoma Picrorhizae is 20～50 weight portions; Flos Caryophylli, Semen Myristicae, the Radix Aucklandiae, Rhizoma Bistortae, Radix Glehniae are 10～30 weight portions, and Artemisia frigida Willd., Lignum Santali Albi, Concretio Silicea Bambusae, Resina Liquidambaris, HEIYUNXIANG, dried Cor Leporis, Lignum Aquilariae Resinatum, Flos Inulae, Flos Bombacis Malabarici, artificial Calculus Bovis, Semen Strychni (processed), Radix Aconiti Kusnezoffii Preparata are 2～20 weight portions.
Adopt decoction and alcohol sedimentation technique to obtain compound extract: decocte with water 3 times merges three times extracting solution, the centrifugal deposition of going.It is 50%～90% that supernatant adds ethanol to final concentration, and decompress filter is removed macromole polar fractions such as polysaccharide.
Adopt organic solvent extractionprocess to carry out initial gross separation: filtrating uses ethyl acetate, n-butanol extraction, concentrate drying to get ethyl acetate component, n-butyl alcohol component successively.
Adopt Isolera 4 full-automatic preparative chromatographs fast that ethyl acetate, the n-butanol extraction position section of drawing are separated: chromatographic column is SNAP 100g chromatographic column (39mm * 157mm; Sweden Biotage company); Filler is normal phase column chromatographic silica gel 200～300 orders, and elder generation is with the low pole mobile phase balance cylinder of 2 column volumes before the eluting.Each component is loaded on after with a small amount of silica gel mixed sample in the appearance post, and applied sample amount is 1: 10～1: 15 with the ratio of silica gel.Eluting adopts linear gradient, flow velocity 40ml/min.Elution program is shown in table 1 and table 2.
Table 1 ethyl acetate composition gradient elution program table
Table 2 n-butyl alcohol composition gradient elution program table
Collect the eluent of ethyl acetate component 34min～69min, 93min～105min respectively, the eluent of n-butyl alcohol component 35min～57min, 67min～73min.According to elution order, in the corresponding time, every 27ml is collected as 1 stream part, adjacent stream part is merged with the different of stream part solute concentration according to the peak type, and 2～20 stream parts can be merged into 1 extract, behind the concentrate drying, have both obtained one group of compound extract.
Provided by the present invention, be one group of extract with antioxidant activity, this group extract can be used as antioxidant and is used to process multiple pharmaceutical composition.Any one or several extract of effective dose and pharmaceutically acceptable excipient or carrier are formed in the treatment that this pharmaceutical composition is made by aforesaid method.Any one or several component with above-mentioned compound extract group of the present invention; After adding various adjuvants or pharmaceutically acceptable excipient or carrier required when preparing different dosage form; Drug formulation process with routine can be prepared into any suitable clinical preparation, for example can be injection (powder pin, freeze-dried powder, liquid drugs injection, transfusion etc.), tablet, oral liquid, granule, capsule, soft capsule, drop pill etc.Wherein, described adjuvant can be antioxygen chelating agent, filler, framework material etc.; Described pharmaceutically acceptable carrier is one or more in xylitol, mannitol, lactose, fructose, glucosan, glucose, polyvinylpyrrolidone, low molecular dextran, sodium chloride, calcium gluconate or the calcium phosphate.
This group compound extract comes from the integral body screening to the whole extracts of compound recipe, and the DPPH method is converted into high flux screening model as screening technique.The compound recipe full constituent to the removing activity distribution of DPPH shown in accompanying drawing, with clearance rate greater than 50% extract as having than the active extract of strong anti-oxidation.In-vitro screening shows that the extract group that the present invention makes has stronger antioxidant activity, especially free radical is had scavenging action preferably, and the center divides into groups to divide the ability of removing free radical far above the free radical scavenger Edaravone.Because the excessive generation of free radical is the major reason of multiple disease development, therefore this group extract is with a wide range of applications; Because this group extract comes from the mongolian medicine compound recipe of treating apoplexy, it possibly have more potential clinical value in the treatment of apoplexy and sequela thereof.
Description of drawings
Accompanying drawing is that the lung-pulse looses the compound recipe full constituent to the active scattergram of the removing of DPPH.Abscissa is the compound extract sample number into spectrum, and vertical coordinate is the clearance rate of sample to free radical DPPH, and the activity of 5～No. 18 samples is higher than Edaravone, has the strong anti-oxidation activity.
The specific embodiment
The preparation of embodiment 1 extract of the present invention
Take by weighing Fructus Chebulae 30g～60g; Rhizoma Picrorhizae 20g～50g; Flos Caryophylli, Semen Myristicae, the Radix Aucklandiae, Rhizoma Bistortae, Radix Glehniae are 10g～30g, and Artemisia frigida Willd., Lignum Santali Albi, Concretio Silicea Bambusae, Resina Liquidambaris, HEIYUNXIANG, dried Cor Leporis, Lignum Aquilariae Resinatum, Flos Inulae, Flos Bombacis Malabarici, artificial Calculus Bovis, Semen Strychni (processed), Radix Aconiti Kusnezoffii Preparata are 2g～20g.After mix homogeneously was also pulverized slightly, decocte with water was extracted 3 times, and each 1.5～2 hours, add water 2～4 parts by volume, merge three times extracting solution, the centrifugal deposition of going.Supernatant adds 3 times of amounts of dehydrated alcohol, fully stirs, and decompress filter, the filtrating partial rotation is evaporated to does not have the alcohol flavor.Ethyl acetate extraction 5 times, medicinal liquid and ethyl acetate volume ratio are 1: 1, combining extraction liquid, concentrate drying get the ethyl acetate component.N-butanol extraction 6 times, medicinal liquid with just be 1: 1 in pure volume ratio.Combining extraction liquid, concentrate drying get the n-butyl alcohol component.
Adopt Isolera 4 full-automatic preparative chromatographs fast, separating ethyl acetate component, n-butyl alcohol component.Chromatographic column is SNAP100g chromatographic column (39mm * 157mm, a Sweden Biotage company), and filler is normal phase column chromatographic silica gel 200～300 orders, and elder generation is with the low pole mobile phase balance cylinder of 2 column volumes before the eluting.Each component is loaded on after with a small amount of silica gel mixed sample in the appearance post, and applied sample amount is 8g.Eluting adopts linear gradient, flow velocity 40ml/min.Elution program is shown in table 1 and table 2.According to elution order, in the corresponding time, every 27ml is collected as 1 stream part.
Table 1 ethyl acetate composition gradient elution program table
Table 2 n-butyl alcohol composition gradient elution program table
The ethyl acetate component is collected the eluent of 34min～69min, and every adjacent 3 stream parts are merged into 1 part; Collect the eluent of 93min～105min, every adjacent 15 stream parts are merged into 1 part.
The n-butyl alcohol component is collected the eluent of 35min～57min, 67min～73min, and every adjacent 4 stream parts are merged into 1 part.
The elute soln of more than collecting is concentrate drying respectively, promptly obtains mongolian medicine compound extract group.
Embodiment 2 extract groups of the present invention are removed the activity rating of free radical
1 instrument and material:
The multi-functional automatic screening appearance of Flexstation3 (U.S. Molecular Devices company), DPPH (SIGMA), dehydrated alcohol (analytical pure), normal saline, dimethyl sulfoxide (analytical pure).
2 experimental techniques:
Hexichol is a kind of more stable fat free love base for bitterness diazanyl free radical (DPPH); Its molecule carries a free electron, and alcoholic solution is purple, and maximum absworption peak is arranged at the 517nm place; Antioxidant can match with the free electron of DPPH; Purple is disappeared, and the absorption value at 517nm place descends, and its decline degree becomes quantitative relationship with the electron number of its acceptance.Principle is with the variation of spectrophotometer detection DPPH free radical and test liquid reaction back light absorption value according to this, and can examine and determine sample provides hydrogen atom, removes free radical antioxidative ability.
The extract sample sets is dissolved in dimethyl sulfoxide, is mixed with the storing solution of 100mg/ml, move into 96 orifice plates, 10 times of normal saline dilutions are diluted three times successively, obtain three gradient concentrations, supply the screening sampling to use.-20 ℃ of freezing preservations
Be reflected in 96 orifice plates and carry out.Each hole adds dehydrated alcohol 160 μ l, and sample well adds sample solution 20 μ l, and control wells adds blank solvent 20 μ l, and its dimethyl sulfoxide final concentration is identical with sample well, measures light absorption value, absorbs as background.Each hole adds DPPH solution 20 μ l, measures light absorption value behind the reaction 30min, and sample well is designated as A behind the deduction background
Sample, control wells is designated as A
ContrastThe clearance rate computing formula is: sample is to DPPH clearance rate (%)=(A
Contrast-A
Sample)/A
Contrast* 100%.Reaction system is 200 μ l, wherein contains ethanol 90%, normal saline 10%, DPPH final concentration 100 μ M.The mensuration wavelength is 521nm.The blank solvent absorption value of selecting not add DPPH is as minimum signal value (minimum signal), and the absorption value behind the blank solvent adding DPPH 30min is as maximum signal level (maximum signal), and imposite is measured Z ' factor values.Computing formula is: Z '=1-3 * (| SD
Maximum Signal|+| SD
Minimum signal|)/(| Mean
Maximum signal|-Mean
Minimum signal|).Extract group to embodiment 1 makes is carried out activity rating.Multiple hole is not established in experiment, repeats 3 times.
3 experimental results:
Z ' the factor of body series is 0.89, and greater than 0.5, screening model is reliable and stable, can be used for screening.
This experimental selection Edaravone is as positive drug.Edaravone is a kind of free radical scavenger of new listing; The excessive free radicals that neurocyte produces during to cerebral ischemia has scavenging action; And the inhibition lipid peroxidation, thereby effective neuroprotective cell, this medicine treatment brain during acute apoplexy of clinical research confirmation is safe and effective.In the neuroprotective research field, the suitable positive drug of this medicine as the free radical scavenger screening model.
With Edaravone solution doubling dilution is 8 concentration; React with DPPH solution respectively, adopt the multi-functional automatic screening appearance of Flexstation3 to measure absorbance, calculate the clearance rate of the Edaravone of variable concentrations DPPH; As shown in table 3, the IC of employing SPSS17.0 computed in software Edaravone
50Value is 4.704.Adopt 2 times of IC
50The approximation 10 μ g/ml of value for screening with the high concentration of positive drug, in the positive drug, low concentration is made as 5 μ g/ml, 2.5 μ g/ml respectively, wait to sieve that sample is also corresponding establishes high, medium and low three concentration.The extract group is as shown in table 4 to the clearance rate of DPPH.
Table 3 variable concentrations Edaravone is to the clearance rate of DPPH
Table 4 extract group is to the clearance rate of DPPH
This group extract has higher removing active to external free radical.Sample segment has still kept higher activity when low concentration, when the 2.5 μ g/ml, free radical scavenging activity is still more than 80%, far above the antioxidant activity level of Edaravone like 5, No. 6 components.
Claims (4)
1. one group of the lung-pulse compound extract that looses is characterized in that being described by its preparation method:
Step 1: take by weighing medical material by the diffusing compound recipe proportioning of the lung-pulse; The medical material proportioning is Fructus Chebulae's 30～60 weight portions; Rhizoma Picrorhizae 20～50 weight portions; Flos Caryophylli, Semen Myristicae, the Radix Aucklandiae, Rhizoma Bistortae, Radix Glehniae are 10～30 weight portions, and Artemisia frigida Willd., Lignum Santali Albi, Concretio Silicea Bambusae, Resina Liquidambaris, HEIYUNXIANG, dried Cor Leporis, Lignum Aquilariae Resinatum, Flos Inulae, Flos Bombacis Malabarici, artificial Calculus Bovis, Semen Strychni (processed), Radix Aconiti Kusnezoffii Preparata are 2～20 weight portions;
Step 2: adopt decoction and alcohol sedimentation technique to obtain compound extract, alcoholic acid final concentration is 50%～90%;
Step 3: use ethyl acetate, n-butanol extraction successively, obtain two extract parts;
Step 4: adopt Isolera 4 full-automatic preparative chromatographs fast that ethyl acetate, the n-butanol extraction position section of drawing are separated; Obtain the eluent of special time period, make the diffusing compound extract group of the lung-pulse, the chromatographic column of using in the step 4 is SNAP 100g chromatographic column; Filler is normal phase column chromatographic silica gel 200～300 orders; Applied sample amount is 1: 10～1: 15 with the ratio of silica gel, and flow velocity is 40ml/min, and it is following that the ethyl acetate extract section of drawing is separated elution program:
Collect eluent at time period 34min～69min, 93min～105min; It is following that the n-butanol portion section of drawing is separated elution program:
Collect eluent at time period 35min～57min, 67min～73min.
2. extract according to claim 1 is characterized in that it being any one section extract of random time collection in the said collection time period of claim 1 or the compositions of several sections extracts.
3. a pharmaceutical composition is characterized in that, said compositions is made up of each described extract and acceptable auxiliary, excipient and additive in the claim 1 and 2 of at least a treatment effective dose.
4. has the application in the medicine of antioxidant activity according to claim 1 or 2 said extracts in preparation.
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| CN2010105333065A CN101979021B (en) | 2010-11-05 | 2010-11-05 | Compound extractive with antioxidation activity |
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| CN2010105333065A CN101979021B (en) | 2010-11-05 | 2010-11-05 | Compound extractive with antioxidation activity |
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| CN101979021B true CN101979021B (en) | 2012-05-30 |
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| CN105232590A (en) * | 2015-03-05 | 2016-01-13 | 刘庆山 | Myrobalan ethanol extract with activity of promoting neural stem cell proliferation and application of myrobalan ethanol extract |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1304769A (en) * | 2001-01-21 | 2001-07-25 | 白玛加措 | Blood-cleaning pill |
| CN1313115A (en) * | 2001-01-21 | 2001-09-19 | 白玛加措 | Medicinal pill for taking care of heart health |
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2010
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1304769A (en) * | 2001-01-21 | 2001-07-25 | 白玛加措 | Blood-cleaning pill |
| CN1313115A (en) * | 2001-01-21 | 2001-09-19 | 白玛加措 | Medicinal pill for taking care of heart health |
Non-Patent Citations (3)
| Title |
|---|
| 刘庆山 等.应用快速色谱技术构建少数民族大复方组分库的方法学研究.《国际药学研究杂志》.2010,第37卷(第4期),310-314. * |
| 张梓倩 等.蒙药白脉散全组分的制备与清除自由基有效成分组的研究.《新药研发暨新药发现学术研讨会会议论文集》.2010,56. * |
| 罗玲 等.补心宝.《中国少数民族传统医药大系》.2000,338. * |
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Owner name: CENTRAL UNIVERSITY FOR NATIONALITIES Free format text: FORMER OWNER: LIU QINGSHAN Effective date: 20121115 |
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Effective date of registration: 20121115 Address after: 100081 Beijing, Zhongguancun, South Street, No. 27, No. Patentee after: Central University for Nationalities Address before: 100081 Institute of traditional medicine, Minzu University of China, Beijing, Haidian District Patentee before: Liu Qingshan |
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Granted publication date: 20120530 Termination date: 20121105 |