CN104706683B - Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes and preparation method thereof and its detection method - Google Patents
Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes and preparation method thereof and its detection method Download PDFInfo
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- CN104706683B CN104706683B CN201310672872.8A CN201310672872A CN104706683B CN 104706683 B CN104706683 B CN 104706683B CN 201310672872 A CN201310672872 A CN 201310672872A CN 104706683 B CN104706683 B CN 104706683B
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- total flavonoids
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Abstract
The invention discloses a kind of Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes and preparation method thereof and its detection method, calculate according to the mass fraction, including 45 55 parts of Process of Total Flavonoids in Drynaria Fortunei, 90 110 parts of natural phosphatidyl cholines and 15 25 parts of cholesterol.Present invention selection Chinese medicine rhizome of davallia extraction Process of Total Flavonoids in Drynaria Fortunei, and Process of Total Flavonoids in Drynaria Fortunei, natural phosphatidyl choline and cholesterol are pressed into certain mass ratio, Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes are prepared under given conditions, and the preparation method is simple and direct, easily operated, convenient control；Prepared rhizome of davallia phospholipid complexes may strengthen the absorption of the medicine, increase dose-effect relationship, improve bioavilability, easy to clinical application efficient extensively, be worth further further investigation.
Description
Technical field
The present invention relates to pharmaceutical field, especially a kind of Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes and preparation method thereof and its inspection
Survey method.
Background technology
The Chinese medicine rhizome of davallia plants the dry root of Mongolian oak fern [Drynaria fortunei (Kunze J.Sm.)] from fire hose section
Stem.Warm-natured, bitter, returns liver and kidney channel, The strong bone of kidney tonifying, cures the wound and relieve the pain；External application disappears wind nti-freckle.Clinically it is more rhizome of davallia medicinal material and
Its preparation is used for set a broken bone, osteoporosis, neck-shoulder pain, lumbago and leg pain, acute or chronic soft tissue bruise, Hiccough and deaf, tooth mobility
Deng.
China's patients with osteoporosis increases year by year at present, seriously affects life of elderly person quality, therefore prevents sclerotin and dredge
Pine is by as the urgent task of medical personal.Traditional Chinese medicine thinks " Shen controls bone, store essential substances, raw marrow ", i.e., kidney essense, which has, promotes bone
The effect of marrow growth, development, reparation.The rhizome of davallia has the effect of typical " The strong bone of kidney tonifying ", therefore in recent years to the Chinese medicine rhizome of davallia
Research is especially active, and pharmacological research shows that the rhizome of davallia can stimulate cartilage cell's compensatory hypertrophy, improves cartilage cell's function, enhancing
The activity of osteoblast, improves bone density, maintains the integrated degree of bone micro-structure, can postpone retrogression pathological changes, reduce Bones and joints
Sick incidence.Pharmaceutical researchers make great efforts to determine that the effective active components of the rhizome of davallia and corresponding definite curative effect, the rhizome of davallia are always yellow
Ketone is exactly the product under this overall background.Research shows in Rhizoma Drynariae extract that main active is Process of Total Flavonoids in Drynaria Fortunei, bone
Broken benefit general flavone, which has, promotes a variety of significant pharmacology such as the healing of bone damage, prevention osteoporosis, anti-inflammatory, antiviral, anti-oxidant to live
Property；Studying the mechanism of Process of Total Flavonoids in Drynaria Fortunei treatment osteoporosis confirms that Process of Total Flavonoids in Drynaria Fortunei can reliably improve bone mineral
Content, these components can help the healing of bone fracture cell, have higher value of exploiting and utilizing.
The rhizome of davallia is more for many years is used for clinic with medicine materical crude slice, therefore the secondary development research of Rhizoma Drynariae extract general flavone has
Wide prospect, by the New technical use in modern pharmacy in the extraction containing drynaria rhizome preparation and preparation process, design
Into more effective new varieties, the clinical application range of this medicinal plant can be extended, so as to expand cultivated area, increases medicinal herb grower
Income.It there is no the research report for preparing Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes both at home and abroad at present.
The content of the invention
The purpose of the present invention is：A kind of Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes and preparation method thereof and its detection side are provided
Method, its preparation method is simple and direct, easily operated, convenient control；Prepared rhizome of davallia phospholipid complexes can strengthen the suction of the medicine
Receive, increase dose-effect relationship, and improve bioavilability, with overcome the deficiencies in the prior art.
What the present invention was realized in：Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes, are calculated according to the mass fraction, including 45-55 parts
Process of Total Flavonoids in Drynaria Fortunei, 90-110 parts of natural phosphatidyl cholines and 15-25 parts of cholesterol.
The preparation method of Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes, Process of Total Flavonoids in Drynaria Fortunei, natural phosphatidyl choline and cholesterol are pressed
Mass fraction is mixed, and adds the chloroform of 15 times of mixture gross mass, under conditions of rotating speed is 4000r/min, magnetic
Power stirs 40min, obtains Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes.
The preparation of the Process of Total Flavonoids in Drynaria Fortunei is that the 8 times of water measured that the rhizome of davallia will be added to add its quality, decoct extraction 3
It is secondary, during decoction, boiling is heated water to, and in order to reduce the evaporation of water, using reflux, 40 minutes every time, it is broken to obtain bone
Mend general flavone.
Naringin content detection method in Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes, using high-efficiency liquid chromatography method for detecting
Detect the naringin content in above-mentioned Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes.
In order to verify the technique effect of the present invention, following experiment has been carried out：
1 experiment material
1.1 reagent sand Rhizoma drynariae preparata (Chongqing Hui Yuan pharmaceutcal corporation, Ltds, lot numbers：20120511)；Aurantiin reference substance (in
Pharmaceutical biological product identification institute of state, lot number：110722-200610, for assay)；Natural phosphatidyl choline (Beijing Suo Laibao science and technology
Co., Ltd, lot number：110511—200912).Cholesterol (Shanghai Blue Season Technology Development Co., Ltd, lot number：20120413)；
Methanol (chromatographically pure, the upper biochemical Co., Ltd of starfish)；Remaining reagent is that analysis is pure.
1.2 instrument Agilent high performance liquid chromatographs (Agilent Technologies1200Series)；Chromatographic column：
Eclipse XDB C18Column (150mm × 4.6mm, 5 μm)；Sartorius BS224S electronic balance (Sai Duolisi scientific instrument
Beijing Co., Ltd)；RE-2000 rotary evaporators (Shanghai Yarong Biochemical Instrument Plant)；SHB-IIIA circulating water types use vacuum
Pump (Zhengzhou Great Wall Trade Co., Ltd.)；98-H-B magnetic agitations electric jacket (Tianjin Stettlen Instrument Ltd.)；KQ-
500DE types numerical control ultrasonic cleaner (Changsha High-Tech Development Zone Hunan instrument Beck instrument finite instrument company)；TU-1901 dual-beams
Ultraviolet specrophotometer (Beijing Puxi General Instrument Co., Ltd)；TGL-16M (16000r/min) desk type high speed freezes
Centrifuge (Community of Jin Tan County Hengfeng Manufacturing Co., Ltd).
2 methods and result
General flavone is isolated and purified after 2.1 experimental designs extraction Process of Total Flavonoids in Drynaria Fortunei, prepares phosphatide complexes, measure phosphatide is answered
Naringin content in compound.
The extraction of 2.2 Process of Total Flavonoids in Drynaria Fortunei and the preparation process of phospholipid complexes
The physics and chemistry of 2.21 rhizomes of davallia, which differentiates, takes a small amount of husky Rhizoma drynariae preparata medicine materical crude slice, uses water to extract and concentrates for (1: 1), then
Qualitive test is carried out to Drynaria Rhizome with the concentrated sulfuric acid, hydrochloric acid-magnesium powder, hydrochloric acid, NaOH solution[7,8,9](being shown in Table 1).
As it can be seen from table 1 strong sulfuric acid response color is changed into aubergine from orange, then illustrate yellow containing double hydrogen in extracting solution
Ketone；And take on a red color with hydrochloric acid-magnesium powder reaction, also illustrate to contain flavanone in extracting solution；Hydrochloric acid is reactionless with extracting solution, says
Chalcone or anthocyanidin are not contained in bright extracting solution.Be in buff for NaOH solution and extracting solution reaction, then explanation extraction
Contain flavonols in liquid.In conclusion the color reaction listed by upper table belongs to the color reaction of flavonoids, therefore show that selected bone is broken
It is correct to mend crude drug, containing Process of Total Flavonoids in Drynaria Fortunei, follow-up test can be carried out.
2.2.2 the extraction experiment of Process of Total Flavonoids in Drynaria Fortunei
Examined or check by identification experiment, amount of water (A), decocting time (B) are set and decoct number (C) 3 factors, Mei Geyin
Element 3 levels (being shown in Table 2) of selection.
2 Process of Total Flavonoids in Drynaria Fortunei extraction factor of tableTable
L is carried out according to table 29(34) orthogonal experiment, each part extracting solution is concentrated to same volume, then measures each group extraction
The content of aurantiin in liquid, so that it is determined that optimal extraction scheme (being shown in Table 3).
3 Process of Total Flavonoids in Drynaria Fortunei orthogonal experiments analytical table directly perceived of table
Note：F0.05(2,2)=19；F0.01(2,2)=99
It is C by the very poor influence size order for understanding each factor in analytical table 3 directly perceived>A>B, can by analysis of variance table 4
Know, only C factors have a significant impact (P to naringin content<0.05), A, B do not make significant difference；Therefore Process of Total Flavonoids in Drynaria Fortunei extraction
Condition is A2B2C3；Each water consumption is selected as 8 times of rhizome of davallia quality, every time 40 minutes, is extracted 3 times.
It is A according to optimum extraction condition2B2C3Larger amount of Process of Total Flavonoids in Drynaria Fortunei is extracted, and concentrates, purify, be freeze-dried,
Prepare to make Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes.
2.2.3L9(34) Process of Total Flavonoids in Drynaria Fortunei phosphatide complexes preparation process orthogonal experiment
Pass through amount (A) of four factors of prerun experimental design for chloroform, Process of Total Flavonoids in Drynaria Fortunei：Natural phosphatidyl choline
(B), mixing time (C), rotating speed (D), each factor set 3 horizontal progress L9(34) orthogonal experiment (being shown in Table 5).
Every part of precision weighs Process of Total Flavonoids in Drynaria Fortunei powder 0.50g (W1) left and right, by L9(34) orthogonal experiment is carried out, prepare phosphorus
Fat body, filtering, drying, weigh the general flavone powder quality (W not wrapped up2), calculate Process of Total Flavonoids in Drynaria Fortunei and phosphatide Percentage bound W
(%)=(W1-W2)/W1× 100% (note：W1For Process of Total Flavonoids in Drynaria Fortunei initial input, W2For Process of Total Flavonoids in Drynaria Fortunei precipitation capacity) meter
Calculate the recombination rate of natural phosphatidyl choline and general flavone.
Note：F0.05(2,2)=19；F0.01(2,2)=99
By Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes prepare in visual result analytical table 6 it is very poor understand each factor influence it is big
Small order is C>D>B>A, show that the scheme for preparing Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes is according to the big I of factor average
A2B3C2D2；From variance table 7, C, D factor have a significant impact (P to the inclusion rate for preparing Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes
<0.05), the influence unobvious of factor A and B, now using the envelop rate of Process of Total Flavonoids in Drynaria Fortunei and natural phosphatidyl choline complex as index,
It is A that can obtain optimal phospholipid complexes preparation process2B3C2D2, i.e. Process of Total Flavonoids in Drynaria Fortunei：When natural phosphatidyl choline is 1: 2, add
15 times of amount solvents, magnetic agitation 40 minutes, rotating speed 4000r.min-1。
2.2.4 Process of Total Flavonoids in Drynaria Fortunei lecithin complex is prepared formula and is determined as by experimental formula by further testing
Process of Total Flavonoids in Drynaria Fortunei：Natural phosphatidyl choline：Cholesterol (5: 10: 2).
2.2.5 the preparation of phospholipid complexes prepares the rhizome of davallia phosphatide of multiple lot numbers according to extraction, the preferred plan prepared
Complex is spare.
2.2.6 marrow mends total flavone phosphatide complex electron microscope and takes the Process of Total Flavonoids in Drynaria Fortunei lecithin complex just prepared
Suspension 1 is dripped on glass slide, and the form of complex is observed in film-making under an electron microscope.
The assay of aurantiin in Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes
2.3.1 chromatographic condition chromatographic column：Eclipse XDB C18Column (150mm × 4.6mm, 5 μm)；Column temperature：25℃；Detection
Wavelength：283nm；Mobile phase is：Methanol: 0.5% glacial acetic acid solution (35: 65)；Flow velocity：1.0mL.min-1；Sample size：20μl.
2.3.2 the preparation of reference substance solution weighs aurantiin standard items 2.5mg according to the preparation precision of product solution, with anhydrous second
Alcohol constant volume is 25mL, and it is 100 μ g.mL that concentration, which is made,-1Aurantiin reference substance solution.
2.3.3 the preparation of test solution takes spare rhizome of davallia phospholipid complexes 1.0g, completely molten with appropriate chloroform
Solution, is demulsified with absolute ethyl alcohol, and whole supernatants are taken out after centrifugation and are settled to 25mL, then accurate measurement 1.00mL with absolute ethyl alcohol again
Into 100ml volumetric flasks, scale is settled to absolute ethyl alcohol, as test solution.
2.3.4 the investigation of linear relationship is accurate respectively measures aurantiin reference substance solution (100 μ g.mL-1) 2.10,2.20,
2.30,2.40,2.50,2.60,2.70mL are settled to scale into 10mL volumetric flasks, with absolute ethyl alcohol, by color under " 2.3.1 " item
Spectral condition is measured, and records chromatogram, is abscissa (X), peak area (S) for ordinate with aurantiin reference substance concentration (C)
(Y) standard curve is drawn, obtains regression equation：Y=18.111X-147.51, r=0.9993.The result shows that aurantiin concentration exists
21.0~27.0 μ g.mL-1It is interior good (being shown in Table 8) with peak area linear relationship.
2.3.5：
The linear relationship data (n=7) of 8 aurantiin of table
2.3.6 precision test draws 24 μ g.mL-120 μ L of aurantiin reference substance solution, repetition sample introduction 6 times, measure is simultaneously
RSD=1.78% of the peak area, as a result aurantiin peak area of aurantiin is recorded, illustrates this method precision preferably (being shown in Table 9).
9 aurantiin reference substance solution Precision test result (n=6) of table
2.3.7 repetitive test takes phospholipid complexes prepared by same batch, by 2.3.3 lower section legal system available test sample solutions
6 parts, 20 μ L injection liquid chromatographs are drawn respectively, and the peak area for being measured by above-mentioned condition and recording aurantiin (is computed sample
Average content be 24.012 μ g.mL-1), as a result RSD=1.92% (being shown in Table 10).
10 sample solution repeated experiment (n=6) of table
2.3.8 stability test takes same batch test solution, respectively in 0,2,4,6,8,10h each 20 μ L of sample introduction, surveys
Determine the peak area of aurantiin, as a result in test sample aurantiin peak area RSD=1.93%, show that test solution is steady in 10h
Qualitative good (being shown in Table 11).
2.3.9 sample recovery rate experiment precision weighs the phospholipid complexes finished product 0.0500g (shaddock peds made from preferred plan
Glycosides content is 60.0300mg.g-1) totally 9 parts, divide 3 groups every group 3 parts, the aurantiin standard items of set component be separately added into each part,
Then it is completely dissolved with chloroform, is demulsified with absolute ethyl alcohol, takes whole supernatants to be settled to again with absolute ethyl alcohol after centrifugation
25mL, then the accurate 1.0mL that measures are settled to scale, by by chromatographic condition under " 23.1 " item into 5ml volumetric flasks with absolute ethyl alcohol
It is measured, and [recovery of standard addition=(the measured value amount-sample theoretical content for adding standard items sample)/standard items as follows
Addition * 100%] rate of recovery is calculated, result of the test illustrates that this method has the preferable rate of recovery (the results are shown in Table 12).
2.3.10 sample size measures
Take the Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes sample of three lot numbers (201211-1,201211-2,201211-3)
Measure wherein aurantiin, its content is 59.7275 respectively, 62.1361,58.2274, RSD (%) be 3.27.
3rd, discuss
Pass through above-mentioned experiment, it is determined that Process of Total Flavonoids in Drynaria Fortunei optimum extraction scheme：Add the water of 8 times of amounts of the rhizome of davallia, decoction carries
Take 3 times, every time 40 minutes；The optimal preparation process that Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes are determined is Process of Total Flavonoids in Drynaria Fortunei：My god
Right lecithin：Cholesterol (5: 10: 2), adds 15 times of chloroform, rotating speed 4000r.min-1, magnetic agitation 40min；Determine
The high-efficiency liquid chromatography method for detecting of aurantiin in phosphatide complexes：Reference substance aurantiin sample size is in 21.0~27.0 μ
g.mL-1Interior have good linear with peak area, Y=18.111X-147.51 (r=0.9993), average recovery rate 99.5%, and RSD is
2.58% (n=9).It is the extraction, preparation process stable operation, reliable, it is easily operated；Naringin content detection method is quick, accurate
Really, it is sensitive, can be as the assay method of aurantiin in Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes.
Modern research shows that the rhizome of davallia can improve bone density, enhancing osteoblast activity, the complete journey for maintaining bone micro-structure
Degree, is one of material base of the kidney generating marrow and dominating bone.Natural phospholipid can be effectively improved with phosphatide complexes made of crude drug component
The absorption of medicine in vivo；Liposome has targeting drug release, slow releasing function, by varying the physicochemical property of active compound thing, strengthens it
Pharmacological action, there is extension action time, significantly improves its biological effectiveness.The present invention is further research Process of Total Flavonoids in Drynaria Fortunei phosphorus
The pharmacological action of fat complex and the novel formulation of development high bioavilability establish certain basis.
By adopting the above-described technical solution, compared with prior art, present invention selection Chinese medicine rhizome of davallia extraction bone is broken
General flavone is mended, and Process of Total Flavonoids in Drynaria Fortunei, natural phosphatidyl choline and cholesterol are pressed into certain mass ratio, is made under given conditions
For into rhizome of davallia total flavone phosphatide complex, the preparation method is simple and direct, easily operated, convenient control；Prepared rhizome of davallia phosphorus
Fat complex may strengthen the absorption of the medicine, increase dose-effect relationship, improve bioavilability, easy to clinical efficient extensively
Using worth further further investigation.The aurantiin of one of main active is answered as the phosphatide using in Process of Total Flavonoids in Drynaria Fortunei
The index components of fit assay, using high performance liquid chromatography as content assaying method, determine the quality of controllable precise
Standard.Rhizome of davallia phospholipid complexes research and develop to the deeply developing of rhizome of davallia medicinal material, rationally using having positive economic and society
Meaning.
Embodiment
The embodiment of the present invention：Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes, are calculated according to the mass fraction, including 45-55 parts of bones are broken
Mend general flavone, 90-110 parts of natural phosphatidyl cholines and 15-25 parts of cholesterol.
The preparation method of Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes, Process of Total Flavonoids in Drynaria Fortunei, natural phosphatidyl choline and cholesterol are pressed
Above-mentioned mass fraction is mixed, and adds the chloroform of 15 times of mixture gross mass, in the condition that rotating speed is 4000r/min
Under, magnetic agitation 40min, obtains Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes.
The preparation of the Process of Total Flavonoids in Drynaria Fortunei is that the 8 times of water measured that the rhizome of davallia will be added to add its quality, decoct extraction 3
It is secondary, during decoction, boiling is heated water to, and in order to reduce the evaporation of water, using reflux, 40 minutes every time, it is broken to obtain bone
Mend general flavone.
Naringin content detection method in Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes, using high-efficiency liquid chromatography method for detecting
Detect the naringin content in above-mentioned Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes.
Claims (3)
- A kind of 1. Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes, it is characterised in that：Calculate according to the mass fraction, including the 45-55 parts of rhizomes of davallia General flavone, 90-110 parts of natural phosphatidyl cholines and 15-25 parts of cholesterol；Its preparation method is：By Process of Total Flavonoids in Drynaria Fortunei, natural ovum Phosphatide and cholesterol are mixed by above-mentioned mass fraction, are added the chloroform of 15 times of mixture gross mass, are in rotating speed Under conditions of 4000r/min, magnetic agitation 40min, obtains Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes.
- A kind of 2. preparation method of Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes as claimed in claim 1, it is characterised in that：Bone is broken Mend general flavone, natural phosphatidyl choline and cholesterol to be mixed by above-mentioned mass fraction, add the trichlorine of 15 times of mixture gross mass Methane, under conditions of rotating speed is 4000r/min, magnetic agitation 40min, obtains Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes.
- 3. the preparation method of Process of Total Flavonoids in Drynaria Fortunei phospholipid complexes according to claim 2, it is characterised in that：The bone The broken preparation for mending general flavone is that the rhizome of davallia is added to the water of 8 times of amounts of its quality, decocts extraction 3 times, 40 minutes every time, obtains Process of Total Flavonoids in Drynaria Fortunei.
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| CN105300903B (en) * | 2015-10-28 | 2018-02-23 | 中国医学科学院输血研究所 | The assay method of total phospholipids content in a kind of hemoglobin oxygen carrier medicine |
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| CN1795927A (en) * | 2004-12-30 | 2006-07-05 | 天津中新药业集团股份有限公司 | Composite of total flavone phospholipid of safflower, and preparation method |
| CN1969871A (en) * | 2005-11-24 | 2007-05-30 | 北京奇源益德药物研究所 | Antivirus compound formulation, preparation process, quality control method and use thereof |
| CN101485722A (en) * | 2009-02-13 | 2009-07-22 | 南京师范大学 | Medicament composition containing drynaria and salvia root, and use thereof |
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2013
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1795927A (en) * | 2004-12-30 | 2006-07-05 | 天津中新药业集团股份有限公司 | Composite of total flavone phospholipid of safflower, and preparation method |
| CN1969871A (en) * | 2005-11-24 | 2007-05-30 | 北京奇源益德药物研究所 | Antivirus compound formulation, preparation process, quality control method and use thereof |
| CN101485722A (en) * | 2009-02-13 | 2009-07-22 | 南京师范大学 | Medicament composition containing drynaria and salvia root, and use thereof |
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