CN108159109A - Radix Astragali flavone extract is preparing the application in treating Neuroleptic Leukocytopenia disease drug - Google Patents
Radix Astragali flavone extract is preparing the application in treating Neuroleptic Leukocytopenia disease drug Download PDFInfo
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- A61K36/185—Magnoliopsida (dicotyledons)
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Abstract
The present invention provides Radix Astragali flavone extract and is preparing the application in treating Neuroleptic Leukocytopenia disease drug, the flavones content of the Radix Astragali flavone extract is 40 60%, main chemical compositions include onocerin, ononin, calycosin, 7 O β D glucopyranoses of calycosin, 7,2 ' dihydroxy 3 ', 4 ' dimethoxy isoflavans, 2 ' hydroxyls 3 ', 4 ' dimethoxy isoflavan, 7 O β D glucopyranoses, 3 hydroxyls 9,10 dimethoxy red sandalwood alkane and 9,10 dimethoxy red sandalwood alkane, 3 O β D glucopyranoses.The Radix Astragali flavone extract of the present invention has the function of preferable increasing leukocyte, and leukopenia or chemicotherapy ancillary drug are treated or prevented available for preparing.
Description
Technical field
The present invention relates to the new application of Radix Astragali flavone extract, more particularly to Radix Astragali flavone extract is preparing treatment or pre-
Application in anti-Neuroleptic Leukocytopenia disease drug.
Background technology
Leukopenia refers to leukocyte count in peripheral blood and is less than 4.0 × 109/L.Leukocyte count reduction will weaken people
Often there is serious infection and bleeding in body immunocompetence, clinic.The reason of leukopenia is fallen ill is more, and such as massive haemorrhage swells
Knurl chemicotherapy, the infection etc. of bacterial virus, but more is as the leukopenia caused by radiation and chemotherapy.
The drug (referred to as rising baiyao) of increasing leukocyte is that leukocyte count, effectively treatment leucocyte subtract in a kind of energy lifting body
The drug of few disease.The liter baiyao clinically used mainly includes Western medicine, biological products and Chinese medicine three categories.Western medicine mainly favorably may be used
Monarch (leucogen), berbamine, vitamin B4, batyl alcohol (α-positive 18 carbon glycerin ether), inosine (inosine), carbonic acid
Lithium, Norcantharidin, caffeic acid etc., but Western medicine can be generated inevitably other while leukopenia is treated
Some side effects limit application clinically；Biological products mainly have Filgrastim (G-CSF), again
Group Filgrastim (rhG-CSF), granular leukocyte macrophage stimulus factor (GM-CSF) and polyethylene glycol recombination
Filgrastim (PEG-rhG-CSF), although the effect of biological products increasing leukocyte is strong, and rapid,
Serious side effect is also resulted in when clinically applied to the leukopenia caused by chemotherapy of tumors and radiotherapy, including serious
Muscle and bone conditions, indigestion, generate heat, headache, fash in addition shock etc..
Radix Astragali is one of parts of generic medicinal plants, first recorded in《Sheng Nong's herbal classic》, there is invigorating qi for strengthening superficies, diuresis, pus draining and toxin expelling is held back
Sore myogenic and other effects.Document report Radix Astragali (Xu Qine etc., Chinese combination of Chinese tradiational and Western medicine magazine, 1995,236-238,395-396；It cares for
Scrupulously and respectfully wave etc., Liaoning Journal of Traditional Chinese Medicine, 2012,39 (11)：2326-2329；Zhou Peifu etc., CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2001,26 (3)：
And astragalus injection (Peng Jiaming etc., Chinese primary health care, 2006,20 (8) 215)：79；Qin Haozhi etc., medical information,
2011,24 (1)：151) there is increasing leukocyte.
The flavonoid glycoside ingredient ononin that Chinese patent CN 104107184A are reported in Radix Astragali has vasodilative
Effect；Chinese patent CN 101596185A, CN 102100691 A, CN 102552241 A, CN 103565790 A, CN
103565791 A, CN 103565793 A, CN 103585143 etc. report onocerin with vasodilator, reduce blood pressure
Effect, inhibit the effect of complement system；Effect to respiratory system；Antifungic action；Anti-allergic effects；Antiviral work
With；104,107,181 104107182 A, CN 1403452 of A, CN of Chinese patent CN, 101590072 A of CN are reported respectively
Immunosupress and anti-inflammatory effect, immunosupress and vasorelaxation action, the anti-heart of calycosin -7-O- β-D- glucopyranoses
Myocardial ischemia effect anti-AIDS effect；101,653,438 104013637 A, CN 1403452 of A, CN of Chinese patent CN,
104107175A, CN 104107181 A, CN 102657641 A, CN 105232522 A report calycosin respectively and exist
The effect of effect, promoting bone growing in terms of cardiovascular system, function of resisting myocardial ischemia, immunosuppressive action, anti-angiogenic property are crazy about
Slow-witted effect, anti-allergic effects；104107182 A of Chinese patent CN report 2 '-hydroxyl -3 ', 4 '-dimethoxy isoflavan -7-
The immunosupress of O- β-D- glucopyranoses and vasorelaxation action；104107183 A, CN 104510752 of Chinese patent CN
A reports the immunosuppressive action of 9,10- dimethoxy red sandalwood alkane -3-O- β-D- glucopyranoses；Prevent Parkinson's disease
Effect.
At present, though Radix Astragali and astragalus injection have function of increasing leukocyte, its active ingredient is unclear, and drug effect is made
With limited, and Radix Astragali flavone constituents and its extract there is no to be reported for the dependent interaction of leukopenia, with above-mentioned
The extract of active ingredient composition also has no its report about treatment leukopenia.
Invention content
The purpose of the present invention is to provide Radix Astragali flavone extracts to prepare treatment or prevention leukopenia or putting
Treat the application in ancillary drug.
Radix Astragali flavone extract provided by the present invention is preparing the application in treating Neuroleptic Leukocytopenia disease drug, described
Radix Astragali flavone extract is prepared by method comprising the following steps：
A. take Milkvetch Root appropriate, the 0-95% ethyl alcohol heating and refluxing extraction that addition liquid-solid ratio is 6-8L/Kg 1-3 times, often
It is 1-3 hours secondary, the extracting solution of gained is merged, paste is concentrated under reduced pressure to obtain；
B. above-mentioned paste AB-8 large pore resin absorption columns are detached, successively with water, 10%, 35% ethanol elution, received
Collect 35% ethanol eluate, it is dry to the greatest extent that eluent is concentrated under reduced pressure into solvent；
C. above-mentioned 35% ethanol eluate concentrate is taken to be scattered in suitable quantity of water, adds the ethyl acetate extraction of 1-3 times of volume
1-2 times, medicinal extract is concentrated under reduced pressure to obtain in combining extraction liquid, and 60 DEG C are dried in vacuo into dry powder.
According to the method described above prepared Radix Astragali flavone extract flavone content be 40-60%, contained primary chemical
Ingredient includes onocerin (1), ononin (2), calycosin (3), calycosin -7-O- β-D- glucopyranoses
(4), 7,2 '-dihydroxy -3 ', 4 '-dimethoxy isoflavan (5), 2 '-hydroxyl -3 ', 4 '-dimethoxy isoflavan -7-O- β-D-
Glucopyranose (6), 3- hydroxyl -9,10- dimethoxy red sandalwood alkane (7), 9,10- dimethoxy red sandalwood alkane -3-O- β-D- pyrans
Glucose (8), structure is as follows：
Pharmacodynamic experiment is the results show that Radix Astragali flavone extract prepared by the present invention can significantly raised leukopenia mould
The quantity of leucocyte of type mouse, effect is significantly better than astragalus injection, and extraction process is simple, of low cost, available for making
It is standby to treat or prevent leukopenia or chemicotherapy ancillary drug.
Description of the drawings
High-efficient liquid phase analysis collection of illustrative plates of the Fig. 1 for Radix Astragali flavone extract, A：Hybrid standard product, B：Radix Astragali flavone extract.
1. Calycosin-7-O-BETA-D-glucoside in figure；2. ononin；3. red sandalwood alkane glycosides；4. Astragaloside-Ⅳ；5. calycosin；6. onocerin；
7. red sandalwood alkane；8. isoflavan.
Specific embodiment
The preparation of 1 Radix Astragali flavone extract of embodiment
The Milkvetch Root of 1000g is taken, after crushing plus water 8L, heating and refluxing extraction 3 times, 1 hour every time, by the extraction of gained
Liquid merges, and paste is concentrated under reduced pressure to obtain.By above-mentioned paste with AB-8 large pore resin absorption columns detach, successively with water, 10%,
35% ethanol elution collects 35% ethanol eluate, and drying is concentrated under reduced pressure, above-mentioned 35% ethanol eluate concentrate is taken to be scattered in
In 200mL water, the ethyl acetate of 1 times of volume is added to extract 2 times, medicinal extract is concentrated under reduced pressure to obtain in combining extraction liquid, and 60 DEG C are dried in vacuo,
Up to Radix Astragali flavone extract 1125mg.
The preparation of 2 Radix Astragali flavone extract of embodiment
The Milkvetch Root of 1000g is taken, adds 20% ethyl alcohol 8L, heating and refluxing extraction 2 times, 2 hours every time, by the extraction of gained
Liquid merges, and paste is concentrated under reduced pressure to obtain.By above-mentioned paste with AB-8 large pore resin absorption columns detach, successively with water, 10%,
35% ethanol elution collects 35% ethanol eluate, and drying is concentrated under reduced pressure, above-mentioned 35% ethanol eluate concentrate is taken to be scattered in
In 200mL water, the ethyl acetate of 2 times of volumes is added to extract 1 time, medicinal extract is concentrated under reduced pressure to obtain in combining extraction liquid, and 60 DEG C are dried in vacuo,
Up to Radix Astragali flavone extract 943mg.
The preparation of 3 Radix Astragali flavone extract of embodiment
The Milkvetch Root of 1000g is taken, adds 95% ethyl alcohol 8L, heating and refluxing extraction 2 times, 1 hour every time, by the extraction of gained
Liquid merges, and paste is concentrated under reduced pressure to obtain.By above-mentioned paste with AB-8 large pore resin absorption columns detach, successively with water, 10%,
35% ethanol elution collects 35% ethanol eluate, and drying is concentrated under reduced pressure, above-mentioned 35% ethanol eluate concentrate is taken to be scattered in
In 200mL water, the ethyl acetate of 2 times of volumes is added to extract 2 times, medicinal extract is concentrated under reduced pressure to obtain in combining extraction liquid, and 60 DEG C are dried in vacuo,
Up to Radix Astragali flavone extract 1064mg.
The chemical composition and content analysis of 4 Radix Astragali flavone extract of embodiment
1st, material：Reference substance：Calycosin -7-O- β-D- glucopyranoses (Calycosin-7-O-BETA-D-glucoside) (lot number：
20120428), 9,10- dimethoxys red sandalwood alkane -3-O- β-D- glucopyranoses (red sandalwood alkane glycosides) (lot number：20120515)、2′-
Hydroxyl -3 ', 4 '-dimethoxy isoflavan -7-O- β-D- glucopyranoses (Astragaloside-Ⅳ) (lot number：20100202), rest-harrow
Plain (lot number：20120528), 3- hydroxyls -9,10- dimethoxy red sandalwoods alkane (red sandalwood alkane) (lot number：20120505), 7,2 '-dihydroxy
Base -3 ', 4 '-dimethoxy isoflavan (isoflavan) (lot number：20120506) Shanghai Yongheng Biological Science and Technology Co., Ltd. is purchased from,
Ononin (lot number：13021808), calycosin (lot number：13082713) Chengdu Man Site companies are purchased from.Reference substance
Purity reaches more than 98%.Deuterated methanol (99.8%, Merck company, Germany), trimethyl silane propionic acid sodium salt (TSP,
Cambridge Isotope Laboratories Inc., MA), ethyl acetate (analyzes pure, Beijing chemical reagents corporation), second
Nitrile (chromatographically pure, Fisher companies, USA).
2nd, instrument：Waters e2695 high-pressure pumps, 2689 UV-vis detectors of Waters, UV, visible light spectrophotometric
Instrument (Shanghai Techcomp Instrument Ltd.).
3rd, efficient liquid phase chromatographic analysis
Chromatographic condition uses C18Chromatographic column (250mm × 4.6mm, 5 μm) with acetonitrile (A)-water (B) for mobile phase, elutes item
Part is shown in Table 1.Flow rate of mobile phase is 1.0mL/min, Detection wavelength 230nm.
1 condition of gradient elution of table
The preparation of reference substance solution：It is yellow to weigh Calycosin-7-O-BETA-D-glucoside, ononin, red sandalwood alkane glycosides, Astragaloside-Ⅳ, Mao Ruiyi
Ketone, onocerin, red sandalwood alkane, isoflavan, 8 reference substances are appropriate, dissolved with chromatography methanol, it is respectively 56.48 μ g/ to be made into concentration
ML, 81.28 μ g/mL, 75.48 μ g/mL, 86.70 μ g/mL, 9.808 μ g/mL, 8.352 μ g/mL, 24.52 μ g/mL and 83.68 μ
The hybrid standard product solution of g/mL, it is spare.
The preparation of test solution：Chromocor extract about 10mg is taken, it is accurately weighed, it puts in 10mL volumetric flasks, adds methanol extremely
Scale shakes up, make by 0.22 μm of miillpore filter to get.
4th, determined by ultraviolet spectrophotometry general flavone
The preparation of Calycosin-7-O-BETA-D-glucoside reference substance series concentration：Calycosin-7-O-BETA-D-glucoside reference substance is taken, is made into concentration point successively
Not Wei 1.086,2.172,4.344,8.688,17.376 μ g/mL reference substance solution.
The preparation of test solution：Radix Astragali flavone extract about 10mg is taken, it is accurately weighed, it puts in 10mL volumetric flasks, adds first
Alcohol to scale, shake up to get.
The selection of spectral conditions：Take above-mentioned Calycosin-7-O-BETA-D-glucoside reference substance solution and Radix Astragali flavone extract test sample molten
Liquid, in carrying out spectral scan in 200~400nm wave-length coverages, as a result its maximum absorption wavelength is in 260nm.It is it is thus determined that purple
Outer Detection wavelength is 260nm.
Using absorbance as ordinate, a concentration of abscissa of Calycosin-7-O-BETA-D-glucoside draws standard curve, obtains regression equation：A
=0.055C, R2=0.9950, illustrate in the range of 1.086~17.376 μ g/mL, Calycosin-7-O-BETA-D-glucoside concentration is in absorbance
Good linear relationship.
5th, Radix Astragali flavone extract chemical composition analysis
As shown in Figure 1：It is compared with reference substance chromatographic peak, there is 8 chromatographic peaks in the HPLC collection of illustrative plates of Radix Astragali flavone extract
Retention time respectively with Calycosin-7-O-BETA-D-glucoside, ononin, red sandalwood alkane glycosides, Astragaloside-Ⅳ, calycosin, onocerin, purple
8 wingceltis alkane, isoflavan flavones ingredients are consistent, illustrate to contain this 8 flavones ingredients in Radix Astragali flavone extract.
6th, in Radix Astragali flavone extract general flavone content analysis
The Radix Astragali flavone extract prepared respectively to above-described embodiment 1-3 using HPLC methods and ultraviolet spectrophotometry (UV)
It is analyzed, the content of general flavone is as shown in table 2, respectively 50%, 46%, 48% (HPLC) and 55%, 50%, 51%
(UV)。
The content of general flavone in 2 Radix Astragali flavone extract of table
Influence of the 5 Radix Astragali flavone extract of embodiment to leukopenia mouse index
1st, material：Kunming mouse, 18~22g of weight are purchased from Military Medical Science Institute's Experimental Animal Center, animal license
Card SCXK (military affairs) 2012-0004.Animal freely ingests, water, is tested after adapting to 1 week.Cyclophosphamide (contains enlightening doctor in Jiangsu
Medicine Co., Ltd, lot number：15092125).The Radix Astragali flavone extract that test medicine is prepared for embodiment 1 is (with sterilizing note
Penetrate and be diluted with water to a concentration of 175 μ g/ml) and astragalus injection (honest QINGCHUN BAO medicine company, lot number：1309133).
2nd, instrument：Animal blood cell analysis machine (HEMAVET 950FS, Drewscientific companies of the U.S.), ultraviolet spectrometry
Photometer (TU-1810, Beijing Puxi General Instrument Co., Ltd), Finland's microplate reader (Labsystems Multiskan
MS, 352 types), Finland's board-washing machine (Thermo Labsystems, AC8), water isolation type constant incubator (GNP-9080 types).
3rd, experimental method
Mouse is weighed, and number is randomly divided into 5 groups, every group of 8 mouse.Respectively normal group, model group, astragalus injection
Group, Radix Astragali flavone extract low dose group and high dose group.It tests the 1st day and starts modeling, model group, astragalus injection group, Huang
Stilbene flavones low dose group and the equal intraperitoneal injection of cyclophosphamide 80mg/kg weight of high dose group mouse, normal group give it is isometric
Physiological saline, continuous injection 3 days；24 hours eye socket 20 μ L of blood sampling after last time injection cyclophosphamide detect the number of haemocyte
Amount；It tests the 4th day, starts intraperitoneal injection and give Radix Astragali flavone extract (low dose group：10mL/kg, high dose group：20mL/
Kg), astragalus injection (dosage：5mL/kg), successive administration 7 days.Posterior orbit blood sampling in 24 hours is administered in Yu Weici, and it is normal to measure blood
Rule；By residual blood in 4 DEG C, 13 000r/min centrifugation 15min, take supernatant, move in EP pipes, for IL-2 in serum,
The measure of IL-6, GM-CSF and HO-1；Mouse is put to death, solution takes mouse left femur, with the CaCl of 0.005mol/L2Rinse bone
In marrow to centrifuge tube, after 4 DEG C are placed 30min, after 2500rpm centrifugations 15min, supernatant is abandoned, with 5mL 0.2mol/L HClO4It is outstanding
It drifts along shallow lake, mixing, after 15min are placed in 90 DEG C of water-baths, cooling, 3500rpm centrifugation 10min take supernatant to measure suction at 268nm
Shading value.
4th, the influence result to blood routine
As shown in Table 3, each group mouse (model group, Radix Astragali flavone extract low dose group, the Radix Astragali Huang of cyclophosphamide are given
Ketone extract high dose group, astragalus injection group) leukocyte count, lymphocyte number, monocyte number and neutrophil leucocyte number it is equal
(compared with blank group, p ＜ 0.01) is remarkably decreased, illustrates the model modeling success of leukopenia.
After administration (experiment the 11st day), compared with model group, Radix Astragali flavone extract low dose group and high dose group, Radix Astragali
Leukocyte count, lymphocyte number, monocyte number, the neutrophil leucocyte number of parenteral solution group mouse have apparent readjustment (p ＜
0.05), i.e. the blood routine of energy increasing leukocyte reduction disease mouse is horizontal, and with the increase of dosage, Radix Astragali flavone carries
Taking the effect of object increasing leukocyte enhances, and the high and low dose group effect of Radix Astragali flavone extract in addition is superior to astragalus injection
(table 4).
Influence (4th day) of the 3 cyclophosphamide modeling of table to mouse blood routine
Note：Compared with normal group, * p ＜ 0.05, * * p ＜ 0.01.
Influence (the 11st day) after the administration of 4 Radix Astragali flavone extract of table to mouse blood routine
Note：Compared with normal group, * p ＜ 0.05, * * p ＜ 0.01；Compared with model group,#P ＜ 0.05,##P ＜ 0.01.
5th, the influence result to marrow DNA content
As can be seen from Table 5, compared with model group, Radix Astragali flavone extract low dose group, the high agent of Radix Astragali flavone extract
Amount group, astragalus injection group have the content of mouse bone marrow cells DNA apparent readjustment effect (p ＜ 0.01), and Radix Astragali flavone extracts
Object is better than astragalus injection to the readjustment effect of mouse bone marrow cells DNA content.
Influence after the administration of 5 Radix Astragali flavone extract of table to mouse bone marrow cells DNA content
Note：Compared with normal group, * p ＜ 0.05, * * p ＜ 0.01；Compared with model group,#P ＜ 0.05,##P ＜ 0.01.
6th, to the influence result of IL-2, IL-6, GM-CSF and HO-1 level in mice serum
By table 6 as it can be seen that the level of IL-2, IL-6, GM-CSF and HO-1 decline in model group mice serum, with normal group
Compare significant difference (p ＜ 0.01).After administration, Radix Astragali flavone extract low dose group, the high agent of Radix Astragali flavone extract
The level of IL-2, IL-6, GM-CSF and HO-1 increase in amount group, astragalus injection group mice serum, compared with model group,
Significant difference (p ＜ 0.01), and Radix Astragali flavone extract makees the readjustment of IL-2, IL-6, GM-CSF and HO-1 in serum
With being better than astragalus injection.
The measurement result of 6 Cytokine of Serum of table
Note：Compared with normal group, * p ＜ 0.05, * * p ＜ 0.01；Compared with model group,#P ＜ 0.05,##P ＜ 0.01.
Claims (3)
1. Radix Astragali flavone extract is preparing the application in treating Neuroleptic Leukocytopenia disease drug.
2. Radix Astragali flavone extract as described in claim 1 is preparing the application in treating Neuroleptic Leukocytopenia disease drug, special
Sign is that the Radix Astragali flavone extract is prepared by method comprising the following steps：
A. take Milkvetch Root appropriate, the 0-95% ethyl alcohol heating and refluxing extraction that addition liquid-solid ratio is 6-8L/Kg 1-3 times, each 1-3
Hour, the extracting solution of gained is merged, paste is concentrated under reduced pressure to obtain；
B. above-mentioned paste AB-8 large pore resin absorption columns are detached, successively with water, 10%, 35% ethanol elution, collected
35% ethanol eluate, it is dry to the greatest extent that eluent is concentrated under reduced pressure into solvent；
C. above-mentioned 35% ethanol eluate concentrate is taken to be scattered in suitable quantity of water, adds the ethyl acetate extraction 1-2 of 1-3 times of volume
Secondary, medicinal extract is concentrated under reduced pressure to obtain in combining extraction liquid, and 60 DEG C are dried in vacuo into dry powder.
3. Radix Astragali flavone extract as claimed in claim 2 is preparing the application in treating Neuroleptic Leukocytopenia disease drug, special
Sign is that the flavones content of the Radix Astragali flavone extract is 40-60%, and contained main chemical compositions include rest-harrow
Element, ononin, calycosin, calycosin -7-O- β-D- glucopyranoses, 7,2 '-dihydroxy -3 ', 4 '-dimethoxy
Base isoflavan, 2 '-hydroxyl -3 ', 4 '-dimethoxy isoflavan -7-O- β-D- glucopyranoses, 3- hydroxyl -9,10- dimethoxys
Red sandalwood alkane, 9,10- dimethoxy red sandalwood alkane -3-O- β-D- glucopyranoses.
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| CN109731031A (en) * | 2019-01-29 | 2019-05-10 | 山西医科大学第一医院 | Application of the Radix Astragali flavone extract in preparation laryngocarcinoma chemotherapeutics |
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| CN101766678A (en) * | 2008-12-26 | 2010-07-07 | 广州康臣药物研究有限公司 | Application of total flavonoid in astragalus to preparing medicaments for preventing and controlling diabetes and nephropathy |
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