CN107281234A - A kind of Hippophate flavone class extract and preparation method thereof - Google Patents
A kind of Hippophate flavone class extract and preparation method thereof Download PDFInfo
- Publication number
- CN107281234A CN107281234A CN201710301922.XA CN201710301922A CN107281234A CN 107281234 A CN107281234 A CN 107281234A CN 201710301922 A CN201710301922 A CN 201710301922A CN 107281234 A CN107281234 A CN 107281234A
- Authority
- CN
- China
- Prior art keywords
- flavone
- ethanol
- extract
- preparation
- hippophate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
Landscapes
- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Alternative & Traditional Medicine (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Medical Informatics (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention provides a kind of Hippophate flavone class extract, general flavone content is 80~95%v/v in the extract.Flavones content (or purity) is significantly higher than commercially available specification in the Hippophate flavone class extract that the present invention is provided, and advantage is provided for the further research of Hippophate flavone；Also provide the higher extraction raw material of purity for the health products or medicine using Hippophate flavone as active component simultaneously.
Description
Technical field
The present invention relates to Hippophate flavone class extract of a kind of high-purity and preparation method thereof.
Background technology
Sea-buckthorn, is the perennial shrub of Elaeangnaceae or magaphanerophytes sea-buckthorn Hippophae rhamnoides Linn. fruit
It is real.Sea-buckthorn is medicinal and edible plant.The root of sea-buckthorn, stem, leaf, flower, fruit, particularly sea buckthorn fruit contain abundant nutriment and
Bioactive substance, can be widely applied to many fields of the national economy such as food, medicine, light industry, space flight, agriculture and animal husbandry fishery.
The effect of sea buckthorn fruit is used as medicine with relieving cough and reducing sputum, stomach strengthening and digestion promoting, promoting blood circulation to remove blood stasis.Modern medicine study, sea-buckthorn can reduce courage and consolidate
Alcohol, allevating angina pectoris breaking-out, also prevents and treats the effect of coronary atherosclerotic heart disease.
It is living containing multivitamin, aliphatic acid, trace element, sub- oily element, Hippophate flavone, superoxides etc. in sea buckthorn fruit
The various amino acid of property material and needed by human body.Research finds that Hippophate flavone has increase coronary blood flow, reduction cardiac muscle
Oxygen demand, suppression platelet aggregation, antitumor, regulation immune system, anti-ageing multiple pharmacological effect of waiting for a long time.
Current commercial Hippophate flavone content (or purity) specification is typically 20%~65%, and content is relatively low, and this is unfavorable
In the further research to Hippophate flavone.
The content of the invention
It is an object of the invention to provide Hippophate flavone class extract of a kind of high-purity and preparation method thereof.
Specifically, the invention provides a kind of Hippophate flavone class extract, in the extract general flavone content be 80~
95%w/w.
Further, general flavone content is 85~94%w/w in the extract；Its content can further reach 89
~94%.For example, in the specific embodiment of the invention, general flavone content can reach about 89% or 94%.
Wherein, the general flavone content, is using rutin as reference substance, using ALCl3Colorimetric method is detected under 270nm
Arrive.
" ALCl of the present invention3Colorimetric method ", is conventional method of this area to determination of total flavonoids, be can refer to general
Operational procedure is carried out.
Present invention also offers the preparation method of above-mentioned Hippophate flavone class extract, it includes following operating procedure：
(1) by sea buckthorn juice, it is freeze-dried to obtain seabuckthorn fruit powder, EtOH Sonicate extracts 2.0~3.0h, ultrasonic power 80~
100W, Extracting temperature is 50~80 DEG C, and volume fraction of ethanol is 40~80%, and solid-liquid ratio is 3~7mL/g, and extraction time is 3
It is secondary；
(2) step (1) extract solution is taken, removes after ethanol, using water as solvent, is divided with styrene tyle macroporous adsorption resin
From, eluted using volume fraction 60-80% ethanol, collect ethanol eluate, remove ethanol, it is dry, produce Hippophate flavone
Class extract.
Further, in step (1), 2.0~3.0h of ultrasonic extraction, ultrasonic power is 100W, and Extracting temperature is 65~75
DEG C, volume fraction of ethanol is 40~60%, and solid-liquid ratio is 5~7mL/g.
Further, in step (1), ultrasonic extraction 2.5h, ultrasonic power is 100W, and Extracting temperature is 65~68 DEG C,
Volume fraction of ethanol is 54~55%, and solid-liquid ratio is 5mL/g.For the ease of operation, in an embodiment of the invention, adopt
Extracting temperature is 65 DEG C, and volume fraction of ethanol is 55%.
Further, in step (2), the styrene tyle macroporous adsorption resin is HPD-100, HPD-826, HPD-600
Or D101.
Further, in step (2), the styrene tyle macroporous adsorption resin is D101.
Further, in step (2), elution volume fraction of ethanol is 80%.
In the case of in view of production cost and efficiency, in step (2), selection uses the mode of Dynamic Adsorption, macropore
The applied sample amount of polymeric adsorbent in 4BV (column volume) below, preferably 4BV；Further, sample solution adsorbs 40~60min at room temperature.
Nitric oxide synthase inhibitors or vascular endothelial growth factor are being prepared present invention also offers above-mentioned total seabuckthorn flavone
Purposes in sub- inhibitor；The nitricoxide synthase is nitric oxide synthase type or/and eNOS.
Heretofore described " removing ethanol ", refers to remove the ethanol in extract solution or eluent as much as possible, wherein
Include the situation for having a small amount of ethanol residual.
Flavones content (or purity) is significantly higher than commercially available specification in the Hippophate flavone class extract that the present invention is provided, and is sea-buckthorn
The further research of flavones provides advantage；Also carried simultaneously for the health products or medicine using Hippophate flavone as active component
The extraction raw material that purity is higher is supplied.
The preparation method of Hippophate flavone class extract of the present invention, only only used relatively conventional alcohol extracting, macroreticular resin pure
Change, without complicated extraction processes such as the sour heavy or supercritical extracts of alkali carries, also involved in again into one not after macroporous resin purification
The means of purification on ground is walked, this seems conventional technology, but can in the short period of time, in specific extraction, purifying bar
Under part coordinates, extreme high purity (more than 80%w/w, it might even be possible to reach 90~Hippophate flavone class 94%) extracts is prepared
Thing, achieves unexpected effect.Meanwhile, acid, alkali may be brought during the inventive method can avoid alkali carries acid heavy
Open loop, closed loop or hydrolysis, can also avoid the high cost of supercritical extract, and the preparation for high-purity Hippophate flavone is provided
A kind of new method of the former flavone component structure of low cost and not malleable.
Result of study of the present invention is also shown that suppression of the extract of the present invention to eNos, iNOS, VEGF etc.
Effect it is more obvious, can also serve as eNos, iNOS or vascular endothelial growth factor receptor inhibitors, treatment eNos, iNOS or
Treatment or the effect of auxiliary treatment are played in the disease (such as atherosclerosis) of vascular endothelial growth factor overexpression.
Obviously, according to the above of the present invention, according to the ordinary technical knowledge and means of this area, this hair is not being departed from
Under the premise of bright above-mentioned basic fundamental thought, the modification of other diversified forms can also be made, replaces or changes.
Below by way of the form of specific embodiment, the above to the present invention is described in further detail again.But no
The scope that this should be interpreted as to above-mentioned theme of the invention is only limitted to following embodiment.It is all to be realized based on the above of the present invention
Technology belong to the scope of the present invention.
Brief description of the drawings
Influence of Fig. 1 difference ultrasonic temperatures to total seabuckthorn flavone acquirement rate
Fig. 2 difference extraction times extract the influence of yield to total seabuckthorn flavone
Fig. 3 different ultrasonic powers extract the influence of yield to total seabuckthorn flavone
Fig. 4 different feed liquids compare the influence that total seabuckthorn flavone extracts yield
Fig. 5 different ethanol concentrations extract the influence of yield to total seabuckthorn flavone
Fig. 6 response surface figures
Five kinds of macroporous absorbent resin adsorption rates of Fig. 7 are calculated
Five kinds of macroporous absorbent resin resolution factors of Fig. 8
Fig. 9 macroporous absorbent resin Static Adsorption kinetic curves
Figure 10 macroporous absorbent resins static state parsing kinetic curve
Figure 11 Dynamic Adsorption curves
Figure 12 dynamic analysis curves
Figure 13 leakage plots
Effect of Figure 14 total seabuckthorn flavones to hyperlipemia rat serum lipids
Effect of Figure 15 total seabuckthorn flavones to hyperlipemia rat liver SOD and MDA
Effect of Figure 16 total seabuckthorn flavones to hyperlipemia rat serum VEGF and VCAM-1
Influence of Figure 17 total seabuckthorn flavones to the effect iNOS and eNOS mRNA level in-sites of hyperlipemia rat
Influence of Figure 18 total seabuckthorn flavones to hyperlipemia rat protein expression
Embodiment
The raw material used for extracting total seabuckthorn flavone of the invention is seabuckthorn fruit powder, is to be freeze-dried gained by sea buckthorn juice.
The preparation of the total seabuckthorn flavone extract of the present invention of embodiment 1
By sea buckthorn juice, seabuckthorn fruit powder is freeze-dried to obtain, EtOH Sonicate extracts 2.5h, and ultrasonic power is 100W, extracts temperature
Spend for 65 DEG C, volume fraction of ethanol is 55%, solid-liquid ratio is 5mL/g, extraction time is 3 times, merges extract solution, removes ethanol
Afterwards, dissolved through distilled water, upper D101 large pore resin absorption columns Dynamic Adsorption, applied sample amount is 4BV, 1h is adsorbed at room temperature, from 60-
80% ethanol is eluted, parsing, is collected eluent, just be can obtain Hippophate flavone class extract of the purity more than 90%.
The conditional filtering of test example preparation method of the present invention
First, the extraction of single factor test combining response face method ultrasonic extraction high-purity total seabuckthorn flavone
The making of 1.1 standard curves：
1.1.1 ALCl is used3Colorimetric method, determine effect preferably, weigh rutin 1.98mg, dissolved with methanol, constant volume in
In 10mL volumetric flask, therefrom draw 5mL, be placed in constant volume in 25mL volumetric flask, then it is accurate draw 1.0mL, 2.0mL,
3.0mL, 4.0mL, 5.0mL and 6.0mL are respectively placed in 10mL volumetric flask, add 5% ALCl3Methanol solution 1mL constant volumes
To 10mL, mix standby
1.1.2 sample treatment：Powdered extract 10mg is taken, is dissolved with methanol solution, is dissolved in surely in 10mL volumetric flask, it is accurate
Measure 2.0mL to be placed in 25mL volumetric flask, add 5% ALCl3Methanol solution 1mL constant volumes, are mixed standby to 10mL
1.2 Detection wavelengths are determined：
Standard items are subjected to full wavelength scanner in 190nm to 600nm, there is absworption peak at 417nm, 270nm, similarly, will
Sample carries out full wavelength scanner in 190nm to 600nm, there is absworption peak at 270nm, therefore is Detection wavelength at selection 270nm, and
It is compared.
The making of 1.3 standard curves：
Standard items are determined into absorbance at 270nm successively, using absorbance as ordinate, standard concentration is abscissa,
Standard curve is drawn, and seeks regression equation.Regression equation：Y=0.0504X+0.0723, R2=0.9998, in 40~240 μ g/
Linear relationship is good within mL.
General flavone recovery rate (%)=(C*V/M) * 100%
V constant volumes, M is that fruit powder quality C is flavones content
2 experiment of single factor results
Influence of 2.1 ultrasonic temperatures to total seabuckthorn flavone recovery rate
Fixed other conditions are constant, (ultrasonic power 100W, solid-liquid ratio is 1 by concentration of alcohol 60%, ultrasonic time 2h:5)
Extract under the extraction conditions of three times, investigate ultrasonic temperature (40,50,60,70,80 DEG C) influence to total seabuckthorn flavone yield.
As a result as shown in figure 1, ultrasonic temperature from 40 DEG C to 70 DEG C when, total seabuckthorn flavone yield gradually rises, ultrasound
When temperature reaches 70 DEG C, the recovery rate of total seabuckthorn flavone can reach highest, be 8.2%, when temperature is 80 DEG C, recovery rate drop
Low, this phenomenon, which is primarily due to flavone compound, has thermal instability, and temperature can decompose it more than 70 DEG C.Therefore,
When ceteris paribus, the optimum temperature of extraction is 70 DEG C.
2.2nd, influence of the ultrasonic time to total seabuckthorn flavone recovery rate
Fixed other conditions are constant, in (liquid ratio 1:5,50 DEG C of ultrasonic temperature, concentration of alcohol 60%, ultrasonic power 100W)
Extract under extraction conditions once, investigate influence of the ultrasonic time (1h, 1.5h, 2h, 2.5h, 3h) to total seabuckthorn flavone yield.
As a result as shown in Fig. 2 the recovery rate of total seabuckthorn flavone is dramatically increased with the increase of ultrasonic time, 2.5h's
When yield be optimal, the yield of total seabuckthorn flavone is more or less the same with 2.5h during 3h, basically identical, it can be seen that, ultrasonic time
2.5h should be selected.
2.3rd, ultrasonic power extracts the influence of yield to total seabuckthorn flavone
Fixed other conditions are constant, in (liquid ratio 1:5,50 DEG C of ultrasonic temperature, concentration of alcohol 60%, ultrasonic time 2h) carry
Take under extraction conditions once, investigate influence of the ultrasonic power (20w, 40w, 60w, 80w, 100w) to total seabuckthorn flavone yield.
As a result as shown in figure 3, total seabuckthorn flavone yield is in power as the increase of ultrasonic power is in significantly rise trend
During 100W, yield is 5.85%.
2.4th, influence of the ultrasonic solid-liquid ratio to total seabuckthorn flavone recovery rate
Fixed other conditions are constant, in (ultrasonic power 100w, 50 DEG C of ultrasonic temperature, concentration of alcohol 60%, ultrasonic time
2h) extract under extraction conditions once, investigate solid-liquid ratio (1:3、1:4、1:5、1:6、1:7) to the shadow of total seabuckthorn flavone yield
Ring.
As a result as shown in figure 4, the yield of total seabuckthorn flavone is in rising trend with the increase of solid-liquid ratio, but one is risen to
When determining degree, ascendant trend slows down, as illustrated, when solid-liquid ratio is 1:When 6, recovery rate is 60%, 1:When 7, recovery rate is
61%, therefore, optimal solid-liquid ratio should select 1:6.
2.5th, influence of the concentration of alcohol to total seabuckthorn flavone recovery rate
Fixed other conditions are constant, in (ultrasonic power 100w, 50 DEG C of ultrasonic temperature, ultrasonic time 2h, solid-liquid ratio 1:5) carry
Take under extraction conditions once, investigate concentration of alcohol (20%, 40%, 60%, 80%, 100%) to total seabuckthorn flavone yield
Influence.
As a result as shown in figure 5, the yield that concentration of alcohol is extracted to total seabuckthorn flavone has important influence, as illustrated, husky
The recovery rate of spine general flavone increases with the increase of concentration of alcohol, when concentration of alcohol increases to 60%, and recovery rate is maximum, when
When concentration is increased to 80% to 100%, recovery rate reduction, this polarity phase with Related Component in the total seabuckthorn flavone with being carried
Close, therefore optimal concentration of alcohol should be 60%.
3 response phase methods are extracted
Screened according to pre-stage test, ultrasonic temperature, ultrasonic time, ultrasonic power and solid-liquid ratio four are mainly studied in this experiment
Influence of the individual factor to response, carries out single factor experiment first, and single factor experiment only investigates Multiple factors individually to response
Influence, it is impossible to reflect the influence of the interphase interaction of Multiple factors；By single factor test trial test, using design-
The softwares of expert 8.0, design the response surface experiments of the level of 4 factor 3, using response surface experiments result, determine total seabuckthorn flavone powder
The optimum extraction condition of extraction.
3.1 response surface experimental results
3.1.1 experimental factor is shown in Table 1 with level design
The center combination design factor of table 1 and water-glass
With A (extraction time), B (Extracting temperature), C (solid-liquid ratio), D (volume fraction of ethanol) for independent variable, sea-buckthorn is always yellow
Ketone content is response (Y), carries out response surface analysis experiment, has 29 experiments, wherein 24 are factorial test, during 5 are
The heart is tested, to evaluated error.29 groups of response surface experiments results, are shown in Table 2.To four argument models, individualism and
The results of analysis of variance under reciprocation, is shown in Table 3.
The response surface experiments result of table 2
The variance point of the fitting quadratic polynomial model of table 3
Note:*, P ＜ 0.01, difference is extremely notable；*, P ＜ 0.05, significant difference.
By carrying out response surface software analysis to result of the test, after Quadratic Regression Fitting, total seabuckthorn flavone yield is obtained
(Y) simulation equation between aforementioned four factor (A, B, C, D) is：Y=8.973+0.159A+0.584B-0.073C+
0.19D-0.248AB+0.039AC+0.066AD+0.01BC+0.606B D-0.132CD-3.02A2-1.58B2-1.82C2-
0.99D2.Regression model F verifies as significantly (P ＜ 0.01), and it, which loses, intends item in the level of α=0.05 not significantly, illustrates the mould
Type is more reliable.Its coefficient of determination R2=0.9972, illustrate that the fit equation is consistent with actual conditions, and error is smaller, can fill
Point reflect the relation between each factor and response, its influence be not be in simple linear relationship.Found by the equation, respectively
It there is certain reciprocation between kind of factor, wherein B, BD, in significantly affecting (P ＜ 0.05), A2、B2、C2、D2It is in pole
Significantly affect (P ＜ 0.01), A is in significantly affecting, and the reciprocation between C, AB, AC, AD, CD, BD is not notable.From single factor test
Level observe, it can be deduced that its influence order be：B (extraction time) ＞ D (volume fraction of ethanol) ＞ A (liquid ratio) ＞ C (are extracted
Temperature).In the presence of reciprocation, the influence order to total seabuckthorn flavone content is：BC ＞ BD ＞ AB ＞ CD ＞ AD ＞ AC.
3.1.2 response surface analysis
Graphics and contour that total seabuckthorn flavone extracts yield response surface are drawn according to model of fit, can intuitively be found out
Influence of the interaction between extreme value and factor to response in the peak of response surface, i.e. parameter area, successively can be with
Three-dimensional response surface and contour map after optimal processing parameter scope, the software processings of Design-expert 8.0 are determined, Fig. 6 is seen.From
Response surface figure (Fig. 6) the observation curve tendency interacted two-by-two between each factor is more smooth, and its influence is smaller；Graph curve
Tendency is steeper, illustrates that its influence is the most notable.Network analysis is carried out by the softwares of Design-Expert 7.0, influence sea-buckthorn is drawn
The optimal extract process of general flavone content is：Liquid ratio 5mL/g, extraction time 2.5h, Extracting temperature are 67.17 DEG C, second
Alcohol volume fraction is 54.88%, and now total seabuckthorn flavone yield estimate is 90.19%.
3.1.3 process certification
Consider the factors such as time, cost, certain amendment has been carried out to technological parameter, it is determined that suitable extraction process
Condition is:Ultrasonic time is 2.5h, and ultrasonic temperature is 65 DEG C, and volume fraction of ethanol is 55%, and solid-liquid ratio is 5mL/g, at this
Under part, it is 9.02% that total seabuckthorn flavone, which averagely extracts yield, is coincide substantially with predicted value.
4 conclusions
The Optimizing Technical of ultrasonic extraction total seabuckthorn flavone is obtained by Box-Behnken center combination experimental designs:
Ultrasonic time is 2.5h, and ultrasonic temperature is 65 DEG C, and volume fraction of ethanol is 55%, and solid-liquid ratio is 5mL/g, and total seabuckthorn flavone is average
It is 9.02% to extract yield.As a result show, the extracting factor for optimizing total seabuckthorn flavone using response phase method has actual answer
With value, theoretical foundation is provided for the further exploitation of total seabuckthorn flavone
2nd, purifying process
HPD-100, HPD-826, HPD-600, D101 are chosen, five kinds of macroporous absorbent resins of AB-8 carry out resin first
Pretreatment, D101, two kinds of resins of AB-8 can soak 24h with ethanol, release immersion liquid, then with distillation water washing, be washed till cleaning solution and add
Water is not muddy, no alcohol taste, then is washed to neutrality with distillation after 5% HCl bubbles 3h, then is washed to neutrality with 2% NaOH bubbles 3h,
It is standby with distilled water immersion.HPD series plastics, wash away tiny resin and broken resin, and ethanol immersion 4h is released and leached
Liquid, add water it is not muddy untill, then with distillation be washed to no alcohol taste, it is stand-by with distilled water immersion.
(1) macroporous absorbent resin adsorption rate is determined
The various resin 1.0g handled well are weighed, surface moisture is blotted with filter paper, is fitted into conical flask with stopper, adds and extracts
Liquid 50mL, sample concentration (C0For 125.733mg/mL), in 25 DEG C of 100r/min constant temperature oscillation 24h, fully filter, survey after absorption
Determine the flavones content in filtrate, and calculate each Adsorbent rate, resolution factor, resin adsorption capacity according to the following formula.
Adsorption rate (%)=(C0﹣ C1)/C0× 100% adsorption capacity (mg/g)=(C0﹣ C1)V/M
C0：Solution flavones initial concentration C before absorption1：Flavones residual concentration in solution after absorption
M：Resin quality V：Flavonoids solution volume
As shown in Figure 7, adsorption rate experiment effect of the five kinds of selected macroporous absorbent resins for total seabuckthorn flavone is tested
It is all relatively good, wherein, the adsorption rate of HPD series and D101 resins is all higher, can reach more than 80%.The absorption of AB-8 resins
Relatively low rate is 75%.
(2) resolution factor of macroporous absorbent resin is determined
Each 1.0g of the saturated resin adsorbed is taken, 50mL (95%) ethanol is added, in 25 DEG C of 100r/min constant temperature oscillators
After 24h, fully parsing, filtering determines the flavones concentration in filtrate, calculates the resolution factor of each resin.Formula：Resolution factor (%)=
C2/(C0﹣ C1) × 100%
C2The concentration of flavones in desorbed solution
As shown in Figure 8, the parsing effect of five kinds of macroporous absorbent resins is all preferable, can reach more than 65%.
(3) the Static Adsorption dynamics of macroporous absorbent resin is determined
According to the comparison of adsorption rate and resolution factor, choose three kinds of resins and carry out adsorption dynamics adsorption kinetics measure, further filter out
Optimum resin, accurately weighs four kinds of each 1.0g of resin, blots surface moisture, be fitted into conical flask with stopper, adds flavonoids solution
50mL, in 25 DEG C of 100r/min constant temperature oscillations, every 0.5h samplings, determines flavones content, calculates each resin adsorption amount, draw quiet
State curve of adsorption kinetics.
As shown above, D101 resins are over time for the Static Adsorption kinetic curve of three kinds of macroporous absorbent resins of selection
Growth, adsorbance gradually increases, and the adsorbance of HPD-100 and HPD-826 resins changes little with the growth of time, reaches
When three hours, the amount of the total seabuckthorn flavone of two kinds of resin adsorptions is substantially close to being significantly less than D101 type macroreticular resins.
(4) Static Adsorption is tested with parsing
Each resin 1.0g is weighed, adds after flavone extractive 50mL is fully adsorbed and filters, take filtrate, flavones concentration is surveyed and calculates
Adsorption rate, to absorption after saturation resin in add various concentrations ethanol solution 50mL, fully parsing after filter, determine parsing
Flavones concentration in liquid, and calculate resolution factor of the ethanol solution to flavones of various concentrations.
As shown in Figure 10, the static parsing effect difference of three kinds of selected macroporous absorbent resins less, parsing amount all with
The increase of concentration of alcohol and increase.Concentration of alcohol resolution factor when 80% reaches maximum, is then sequentially reduced.
(5) Dynamic Adsorption is tested with parsing
1) appropriate macroporous absorbent resin is taken, wet method is fitted into glass column, by concentration on 0.1257mg/mL flavonoids solution
Sample, is adsorbed with 1.0mL/min flow velocitys, efflux is collected, and one is collected per 5mL and is managed, and detects that each flow point sea-buckthorn is total
The concentration of flavones, using effluent volume as abscissa, total seabuckthorn flavone concentration is ordinate, draws adsorption curve.
2) after absorption terminates, with 95% ethanol solution, eluted with 1.0mL/min flow velocitys, one collected per 5mL and is managed,
The concentration of each flow point total seabuckthorn flavone is detected, using effluent volume as abscissa, total seabuckthorn flavone concentration is ordinate, is drawn
Elution curve.
As seen from Figure 11, during absorption, D-101 types resin is in 15min, and adsorption rate is significantly increased, and is reached during 20min
It is then identical with other two kinds of resins to maximum；It is visible according to Figure 12 dynamic analyses experiment diagram, the parsing effect of three kinds of resins
Fruit is changed with time basically identical.
In above-mentioned experiment, D101 macroporous absorbent resins have the efficiency of preferably separation Hippophate flavone class material, therefore, choosing
Take D101 types macroporous absorbent resin further to carry out applied sample amount and repeat experiment and checking test,
(6) investigation of loading mass concentration:
Obtained Hippophate flavone class sample dilution will be extracted, respectively by D101 type macroporous absorbent resins, keep applied sample amount
It is identical with volume flow, influence of the different loading mass concentrations to macroporous absorbent resin adsorption effect is investigated, 4 are the results are shown in Table.
Influence of the different loading mass concentrations of table 4 to adsorption effect
As a result show that different loading mass concentrations influence little to the absorption behavior of D101 type macroporous absorbent resins, therefore choosing
The direct loading of Hippophate flavone extract is selected, is conducive to operation.
(7) drafting of leakage plot:
By above-mentioned identified adsorption conditionses, Dynamic Adsorption, Fractional Collections efflux are carried out.Every part of 10mL, collects 25 parts.
To determine total seabuckthorn flavone mass concentration as inspection target, leakage plot is drawn, Figure 13 is as a result seen.It can be seen that, when loading is big
During about 40~50mL (4~5BV), total seabuckthorn flavone just substantially have leaked, it is thus determined that the sample that maximum applied sample amount is 4BV
Liquid.
(8) resin reuses the investigation of number of times:
According to above-mentioned identified absorption and elution requirement, sample liquid is taken to carry out upper prop, absorption, elution, in same tree
Repeated on fat post 4 times, 4 general flavone adsorbances are calculated respectively, total seabuckthorn flavone adsorbance is bright when as a result the 4th is used
It is aobvious to decline, after illustrating that D101 types macroporous absorbent resin is reused 3 times, need regeneration to be just continuing with.
(9) checking test
According to above-mentioned identified purifying total seabuckthorn flavone absorption and elution requirement, sample liquid is taken to carry out upper prop, adsorb, wash
It is de-, the mass fraction 93.89% of total seabuckthorn flavone, the rate of recovery is 81.11%.It can be seen that, through D101 type macroporous absorbent resins
After purification, the paste-forming rate of total seabuckthorn flavone substantially lowers, and good impurity removing effect, mass fraction reaches more than 90%.
2.3 conclusion
It was found from the result of above-mentioned experiment, HPD-100, HPD-826, HPD-600, D101, five kinds of resins of AB-8 are respectively provided with
Purifying total seabuckthorn flavone effect, by experiment sieving, comprehensive each performance indications and subsequent authentication experiment, final work
Skill flow is：The Hippophate flavone class extract of this experiment extraction process extraction is chosen, is dissolved through distilled water, D101 macroporous absorption trees
Fat post Dynamic Adsorption, maximum applied sample amount is 4BV, and 40min~60min is adsorbed at room temperature, is eluted from 80% ethanol, is solved
Analysis, collects eluent, just can obtain Hippophate flavone class extract of the purity more than 90%.
Mechanism Study of the high-purity Hippophate flavone class extract of the present invention of test example 1 to blood vessel endothelium protection activity
1. materials and methods
1.1 experiment material
1.1.1 experimental animal
4 SPF grades of week old SD hero mouse (quality certification no.62001000000134) 60,90 ± 10g is purchased from Gansu traditional Chinese medicine
University.
1.1.2 instrument and reagent
EYELAN1100 Rotary Evaporators, plum Teller MS105 type electronic balances, HC-3018R refrigerated centrifuges, ELIASA,
Ultraviolet specrophotometer, macroporous absorbent resin, cholesterol, cholate, propylthiouracil, Tween-80, distilled water, physiological saline,
High lipid food (Beijing Austria of section pull together company)；TC, TG, HDL, LDL, SOD, MDA, VEGF, VCAM-1 kit (Nanjing is built up)；
Water-bath, ELIASA, centrifuge, absolute ethyl alcohol (Tianjin all generations Chemical Co., Ltd.)；eNOS、iNOS、LOX-1、ICAM-1、
GAPDH (Abcam), goat-anti rabbit HRP mark secondary antibody, sheep anti mouse HRP mark secondary antibodies (the green skies).
1.1.3 the preparation of total seabuckthorn flavone：
Fresh fructus hippophae, picks up from Qinghai Province's cajaput and Guoluo (96.97 ° of E, 33.03 ° of N, 100.23 ° of E, 34.48 °
N).5kg sea buckthorn juices are freeze-dried, 2kg is obtained and dries fruit powder, with 70% ethanol with solid-liquid ratio 1:3,60 DEG C extract 3 times, often
Secondary two hours, concentration filtrate obtained 1kg, and gained medicinal extract is dissolved in distilled water, separated with D101 macroporous absorbent resins,
50%-70% ethanol is eluted, and is finally obtained refined total seabuckthorn flavone 35g, is measured flavones in final total seabuckthorn flavone
Content is 89.98 ± 0.032%.The general flavone content, is using rutin as reference substance, using ALCl3Colorimetric method is under 270nm
Detection is obtained.
1.2 experimental method
1.2.1 experimental animal modeling and administration
High blood lipid model is set up：Buy after animal, after normal diet is adapted to three days, be randomly divided into Normal group (abbreviation
For NC), model control group (is abbreviated as MC), and positive controls (are abbreviated as PC), (abbreviation of the basic, normal, high dosage group of total seabuckthorn flavone
For VPH, VPM, VPL), Normal group gives basal feed, and remaining gives high lipid food 6 weeks, and in giving fat every other day
Emulsion 4 weeks, is alternately illuminated, high lipid food formula is for 12 hours：12% lard, 10% yolk powder, 5% cholesterol, 1% cholate,
0.2% propylthiouracil, 71.8% basal feed；Fat emulsion formula is：10% lard, 5% cholesterol, 1% cholate,
0.2% propylthiouracil, 1% Tween-80, remaining be distilled water.During modeling, two animals, eye are randomly selected weekly
Bottom venous blood sampling, detects blood lipids index, and checking model sets up situation.Modeling starts administration on the 7th week, and gavage (i.g) give medicine
5 weeks, the high, medium and low dosage group dosage of total seabuckthorn flavone was respectively 7mg/kg, 14mg/kg, 28mg/kg, using simvastatin as the positive
Comparison medicine, dosage is 0.3mg/kg；Give blank control group and model group distilled water simultaneously；During experiment, each group rat is recorded
Diet, drinking-water and body weight.
1.2.2 serum parameters are detected
After gastric infusion 5 weeks, each group Rat Fast is prohibited into water 12 hours, 20% urethane 0.5mL/kg, abdomen is injected intraperitoneally
Sustainer takes blood, and 4000r/min centrifugations take serum.Using TBA colorimetric determination liver MDA (MDA) content, WST-1 methods
Liver Superoxide Dismutase Activity (SOD) vigor of detection；Determine T-CHOL (TC), triglycerides (TG), HDL
And low-density lipoprotein (LDL) (HDL).Detect VEGF (VEGF) and the vascular cell adhesion factor (VCAM-
1)。
1.2.3 rat aorta metamorphosis is observed
Sample is pre-fixed through 3% glutaraldehyde, and 1% osmium tetroxide is fixed again, and acetone is dehydrated step by step, Epon812 embeddings, and half is thin
Section optical alignment, ultra-thin section, acetic acid uranium and lead citrate double staining, Hitachi H-600IV type transmission electron microscope observings.
1.2.4 sustainer eNOS and the detection of iNOS mRNA level in-sites
1.2.4.1 Total RNAs extraction：Experimental animal is taken after blood, is removed aorta pectoralis, is softly separated surrounding tissue, is placed in life
Cleaned up in reason salt solution, be cut into segment, take tissue or cell in 1mL Trizol homogenate tube, in refiner homogenate
20sec, is placed in super-clean bench, incubates 5min, 12000r/min, centrifuges 10min；Suct clearly in new 1.5mL centrifuge tubes, plus
Enter 200 μ L chloroform, shake up, be stored at room temperature 2min, 4 DEG C, 12000r/min centrifuges 10min；Supernatant is drawn in new 1.5mL
In centrifuge tube, 600 μ L isopropanol is added, is well mixed, 15min is stored at room temperature, 4 DEG C, 12000r/min centrifuges 15min, abandoned
Supernatant；Add 1mL75% absolute ethyl alcohol (the μ L DEPC water of 750 μ L absolute ethyl alcohols+250) rinsing precipitation, 4 DEG C, 12000r/
Min, centrifuges 5min, abandons supernatant；1mL absolute ethyl alcohols are added, rinsing is precipitated, 4 DEG C, 12000r/min centrifuges 5min, abandons supernatant,
Drying at room temperature 10min；40 μ L DEPC water dissolving RNA is added, -80 DEG C of refrigerators is placed in and saves backup.
1.2.4.2 the synthesis of first chain of cDNA：The DNA in total serum IgE is eliminated, with reverse transcription reagent box in corresponding system
Middle reaction.
1.2.4.3PCR expand, the cDNA prepared is entered into performing PCR amplification.Primer sequence is as follows：
The RT-PCR primer sequence of table 5
1.2.5 sustainer eNOS and iNOS and the detection of LOX-1, ICAM-1 protein level
By the blood vessel being cut into small pieces, protein lysate cracking tissue, homogenate, centrifugation are added, supernatant is taken, uses BCA albumen
Assay kit detects sample total protein concentration.Extract albumen and be denatured 5min in 100 DEG C with 5 × albumen sample-loading buffer.With
Albumen is separated by electrophoresis in 10%SDS-PAGE, is 80 μ g per swimming lane loading protein content.Transferred with half-dried transfer groove (BioRad, USA)
On albumen to pvdf membrane, 2h is closed at room temperature with 5% skimmed milk power afterwards, is separately added into rabbit-anti eNOS, iNOS, LOX-1,
ICAM-1 rabbit-antis GAPDH (1:1500), in 4 DEG C of overnight incubations.Wash after film and add the goat-anti rabbit through horseradish peroxidase-labeled
Secondary antibody (1:1000) 2h, is incubated at room temperature, afterwards using the luminous development of ECL liquid, after scanning film, using Image J softwares to solidifying
Glue image protein expression band integral optical density value is analyzed, and calculates eNOS, iNOS, LOX-1, ICAM-1 and GAPDH bar
Relative ratio with integral optical density value.
2 experimental results
2.1 experimental rat changes of weight
After modeling, the body weight that high lipid food rat body weight value is significantly higher than rats in normal control group is given, after administration, each
Medicine group body weight increase speed reduce, administration second week medicine group body weight increase be remarkably decreased, total seabuckthorn flavone high dose group and
The rat body weight of middle dose group, which increases, is less than Normal group, significant difference；Illustrate that total seabuckthorn flavone has and suppress body weight increase
Effect, specific result is see table 6.
The influence of the total seabuckthorn flavone hyperlipemia rat body weight of table 6.
Note：Compared with model group,▲P ＜ 0.05,▲▲P ＜ 0.01.
2.2 rat aorta morphologic observation results
In each experimental group rat aorta structure of electric Microscopic observation, wherein, model group rats endothelial cell presents a series of
Abnormal performance, chromatin agregation, some foam cells of reserves, and also eucaryotic cell structure is unintelligible, and there is Endothelial denudation
Phenomenon, with mitochondrial swelling；On smooth muscle cell, some physalises are also observed that.Compared with model group, just
Often the endothelial cell and smooth muscle cell of group rat are barely perceivable above-mentioned situation, and eucaryotic cell structure is completely clear, organelle knot
Structure is normal.In addition, the visible a small amount of foam cells of each dosage group and positive controls of medication, has necessarily compared to model group
Improvement, the low middle dose group and positive controls of total seabuckthorn flavone also show a certain degree of mitochondrial swelling, it is comprehensive each
The Morphological comparison of group, experiment finds the cellular morphology of total seabuckthorn flavone high dose group preferably, can effectively improve suppression high fat of blood
Caused Aortic Morphology change.
Foam cells refers to contain many fat drips in the monocyte or histocyte for having swallowed lipid, its endochylema, is
The characteristic pathological cell occurred in pulse atherosclerosis patch, is mainly derived from blood mononuclear cell and tunicae media vasorum smooth muscle is thin
Born of the same parents.Foam wanshing is the earliest events of atherogenesis.
2.4 Blood Lipid
Figure 14 is shown in influence of the total seabuckthorn flavone to hyperlipemia rat serum lipids.After modeling, model group serum lipids index
Significantly rise, TC, TG and LDL rises, be respectively (2.25 ± 0.2,0.627 ± 0.03,0.399 ± 0.03) mmol/mL,
HDL is reduced, and is worth for 0.486 ± 0.02；After successive administration five weeks, administration group Serum TC, TG, LDL is notable compared with model group
Decline, HDL is significantly raised, and difference has statistical significance；And be in dose-effect relationship between each dosage group；Comparatively speaking,
The effect for reducing blood fat of each dosage group of total seabuckthorn flavone is better than positive controls, in total seabuckthorn flavone high dose group blood lipid level with just
Often organize close.This experimental result illustrates that total seabuckthorn flavone can significantly reduce hyperlipidemia ratses serum TC, TG, tri- indexs of LDL,
And have facilitation to rat blood serum HDL rises.
2.5 liver SODs and MDA assays
Figure 15 is shown in influence of the total seabuckthorn flavone to hyperlipemia rat liver SOD and MDA.As schemed, the SOD reductions of model group
And MDA is raised, by the intervention of total seabuckthorn flavone, the liver SOD and MDA of each dosage group improve, in dose-effect relationship.
2.6 serum VEGFs and VCAM-1 levels
Figure 16 is shown in influence of the total seabuckthorn flavone to hyperlipemia rat serum VCAM-1 and VEGF.As schemed, the VCAM- of model group
1 and VEGF, two indexs are raised, by the intervention of total seabuckthorn flavone, and the VCAM-1 and VEGF of each dosage group are significantly reduced,
In dose-effect relationship.
2.7 Rt-PCR
INOS and eNOS mRNA expressions are detected by RT-PCR, Figure 17 is as a result seen.As a result show, model group is actively
Arteries and veins blood vessel iNOS and eNOS expression is all significantly higher than other each groups, by the dry of total seabuckthorn flavone and positive control medicine
The expression of preact, medicine group and positive group is reduced, and it neutralizes model group and compared, the significant difference of middle high dose group.
2.8 Western blot
Western blot experimental results are shown in Figure 18, iNOS and eNOS protein expression level and mRNA expressions one
Cause.Model group is reduced, and each medicine group expression is raised successively, in dose-effect relationship.
3rd, conclusion
Therefore, this result of study shows compared with normal group, and model group rats blood lipid level is significantly increased, oxidation resistance
Reduction, Aortic injury.And total seabuckthorn flavone can suppress iNOS, eNos, VEGF expression, by adjust eNOS/NO and
INOS/NO paths and oxidation resistance improve the dysfunction of body aorta inner skin and damage caused by long term high-fat diet.
Certainly, extract of the present invention is more obvious to the inhibitory action of eNos, iNOS, VEGF etc., together
Sample can be as eNos, iNOS or vascular endothelial growth factor receptor inhibitors, in treatment eNos, iNOS or VEGF
Treatment or the effect of auxiliary treatment are played in the disease of overexpression.
SEQUENCE LISTING
<110>Northwest Plateau-organisms Research Inst. of Chinese Academy of Sciences
Qinghai University
<120>A kind of Hippophate flavone class extract and preparation method thereof
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213>ENOS gene forward primer sequences
<400> 1
ctttcggaag gcgtttgac 19
<210> 2
<211> 19
<212> DNA
<213>ENOS gene reverse primer sequences
<400> 2
aactcttgtg ctgctcagg 19
<210> 3
<211> 18
<212> DNA
<213>INOS gene forward primer sequences
<400> 3
aggcttgggt cttgttag 18
<210> 4
<211> 18
<212> DNA
<213>INOS gene reverse primer sequences
<400> 4
ttgttgggct gggaatag 18
Claims (10)
1. a kind of Hippophate flavone class extract, it is characterised in that：General flavone content is 80~95%w/w in the extract.
2. Hippophate flavone class extract according to claim 1, it is characterised in that：General flavone content is in the extract
85~94%w/w；It is further 89~94%w/w.
3. Hippophate flavone class extract according to claim 1 or 2, it is characterised in that：The general flavone content, is with reed
Fourth is reference substance, using ALCl3Colorimetric method is detected under 270nm and obtained.
4. the preparation method of Hippophate flavone class extract described in claims 1 to 3 any one, it is characterised in that：It is included such as
Lower operating procedure：
(1) sea buckthorn juice is taken, seabuckthorn fruit powder is freeze-dried to obtain, ultrasonic extraction is carried out by 40~80% ethanol of volume fraction
2.0~3.0h, 80~100W of ultrasonic power, Extracting temperature are 50~80 DEG C, and solid-liquid ratio is 3~7mL/g, is extracted 3 times；
(2) step (1) extract solution is taken, removes after ethanol, using water as solvent, is separated with styrene tyle macroporous adsorption resin,
Eluted using the ethanol of volume fraction 60~80%, collect ethanol eluate, remove ethanol, dried, produce Hippophate flavone class
Extract.
5. preparation method according to claim 4, it is characterised in that：In step (1), 2.0~3.0h of ultrasonic extraction, ultrasound
Power is 100W, and Extracting temperature is 65~75 DEG C, and volume fraction of ethanol is 40~60%, and solid-liquid ratio is 5~7mL/g.
6. preparation method according to claim 5, it is characterised in that：In step (1), ultrasonic extraction 2.5h, ultrasonic power
For 100W, Extracting temperature is 65~68 DEG C, and volume fraction of ethanol is 54~55%, and solid-liquid ratio is 5mL/g.
7. preparation method according to claim 4, it is characterised in that：In step (2), the styrene tyle macroporous absorption tree
Fat is HPD-100, HPD-826, HPD-600 or D101；Preferably use D101.
8. preparation method according to claim 4, it is characterised in that：In step (2), elution is with volume fraction of ethanol
80%.
9. preparation method according to claim 4, it is characterised in that：In step (2), Dynamic Adsorption, macroporous absorption are selected
The applied sample amount of resin is in below 4BV；Further, 40~60min is adsorbed at room temperature.
10. total seabuckthorn flavone described in claims 1 to 3 is preparing nitric oxide synthase inhibitors or VEGF suppression
Purposes in preparation；The nitricoxide synthase is nitric oxide synthase type or/and eNOS.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610436642 | 2016-06-13 | ||
| CN2016104366425 | 2016-06-13 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107281234A true CN107281234A (en) | 2017-10-24 |
Family
ID=60094369
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710301922.XA Pending CN107281234A (en) | 2016-06-13 | 2017-05-02 | A kind of Hippophate flavone class extract and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107281234A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110548053A (en) * | 2018-06-04 | 2019-12-10 | 大兴安岭至臻尚品寒带生物技术有限公司 | a method for preparing fructus Hippophae extract rich in total flavonoids and natural vitamin C |
| CN110643582A (en) * | 2019-09-29 | 2020-01-03 | 浙江工业大学 | Method for extracting SOD from fresh sea-buckthorn fruit |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101559118A (en) * | 2008-04-14 | 2009-10-21 | 北京卓越同创药物研究院 | Medicine composition with blood fat adjusting effect and preparation method thereof |
| CN103054915A (en) * | 2012-12-13 | 2013-04-24 | 大兴安岭林格贝有机食品有限责任公司 | Novel production process for total flavones of hippophae rhamnoides |
-
2017
- 2017-05-02 CN CN201710301922.XA patent/CN107281234A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101559118A (en) * | 2008-04-14 | 2009-10-21 | 北京卓越同创药物研究院 | Medicine composition with blood fat adjusting effect and preparation method thereof |
| CN103054915A (en) * | 2012-12-13 | 2013-04-24 | 大兴安岭林格贝有机食品有限责任公司 | Novel production process for total flavones of hippophae rhamnoides |
Non-Patent Citations (2)
| Title |
|---|
| 姜少娟等: "超声波法提取沙棘果渣中总黄酮的最佳工艺研究", 《西北农林科技大学学报（自然科学版）》 * |
| 杨云裳等: "沙棘汁中总黄酮提取及纯化研究", 《时珍国医国药》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110548053A (en) * | 2018-06-04 | 2019-12-10 | 大兴安岭至臻尚品寒带生物技术有限公司 | a method for preparing fructus Hippophae extract rich in total flavonoids and natural vitamin C |
| CN110643582A (en) * | 2019-09-29 | 2020-01-03 | 浙江工业大学 | Method for extracting SOD from fresh sea-buckthorn fruit |
| CN110643582B (en) * | 2019-09-29 | 2021-07-27 | 浙江工业大学 | Method for extracting SOD from fresh sea-buckthorn fruit |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104825551B (en) | A kind of buckwheat shell chromocor extract and application | |
| CN101828593A (en) | Preparation method of composite green health food of freeze dried purslane and purslane/seaweed | |
| Wang et al. | In vitro and in vivo antioxidant activities of polyphenol extracted from black garlic | |
| CN107281234A (en) | A kind of Hippophate flavone class extract and preparation method thereof | |
| CN107412721A (en) | A kind of hypoglycemic bitter gourd polypeptide compound capsule and preparation method thereof | |
| CN104644621A (en) | Application of chlorogenic acid in preparation of medicines for treating cardiomyopathy | |
| CN104721301B (en) | A kind of apple polyphenol ethanol extract and its preparation method and application | |
| CN103961379B (en) | Laetiporus sulphureus extract and application thereof | |
| CN105125601B (en) | A kind of extract of penthorum chinense pursh and its preparation method and application | |
| CN101278970B (en) | Method for preparing Elsholtzia bodinieri flavonoid and applications | |
| CN104873616B (en) | Application of the litchi rind polyphenol in the medicine or health products that reduce liver tg is prepared | |
| CN103479875B (en) | A kind of preparation method of high activity Rhizoma Polygonati Odorati concentrated solution and application thereof | |
| CN106109668B (en) | Inhibit the purification process of liver cell lipid aggregation main effect phenols component in litchi pulp | |
| CN113730466A (en) | Preparation method and application of blackberry extract | |
| CN104926958A (en) | Method for extracting herba lycopi polysaccharide from herba lycopi and application of herba lycopi polysaccharide | |
| CN103665180A (en) | Application of Morindae officinalis oligosaccharide 6 glycan in preparation of myocardial ischemia and reperfusion injury resistance medicines | |
| CN101757059B (en) | Method for extracting alpha-glucosidase activity inhibitor from plants | |
| CN104606164B (en) | A kind of preparation method of safflower " Jinghuasu " essence capsule | |
| CN103638162A (en) | Application of morindae officinalis oligosaccharide tetrasaccharide to preparation of drug for treating myocardial ischemia and reperfusion injury | |
| CN105520847B (en) | A kind of heal-care essential oil and preparation method thereof containing JINHUAKUIZI oil | |
| CN103319563B (en) | Extraction and purification method of soyasaponins | |
| CN107281233A (en) | Protection purposes of the total seabuckthorn flavone to blood vessel endothelium | |
| CN106942439B (en) | Cyclocarya paliurus and guava leaf blood glucose reducing tea and preparation method and application thereof | |
| CN103638034A (en) | Application of morindae officinalis oligosaccharide pentasaccharide to preparation of drug for treating myocardial ischemia and reperfusion injury | |
| CN104922271B (en) | Application of the litchi rind polyphenol in the medicine or health products that reduce hepatic cholesterol is prepared |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20171024 |