CN106610406A - Micro-extraction method of honeysuckle - Google Patents
Micro-extraction method of honeysuckle Download PDFInfo
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Abstract
The invention discloses a method for extraction and detection of phenolic acid effective components in honeysuckle medicinal materials by using beta-cyclodextrin as an adsorbent for matrix solid-phase dispersion extraction technology. The method includes: adding honeysuckle herbal powder and a beta-cyclodextrin adsorbent into a mortar to conduct grinding, taking a solid-phase extraction small column, adding a sieve plate at the bottom, filling the small column with grinded solid, then adding a sieve plate at the top, placing the filled small column on a solid-phase extraction instrument, taking an eluant and injecting it into the small column, conducting elution on the small column, using a centrifuge tube to collect eluent at the bottom, then conducting volution and centrifugation on the eluent in the centrifuge tube, and taking supernatant liquid, i.e. a honeysuckle phenolic acid effective component extracted liquid. Compared with the traditional extraction method, the method used by the invention has more concise and faster steps, saves time and labour, greatly reduces the organic solvent dosage, has remarkable extraction effect, and can effectively extract phenolic acid components in honeysuckle.
Description
Technical field
The invention belongs to natural drug extracts detection field.It is related to a kind of Trace bio-element method of medicinal plants, specifically
Ground say, it is related to by beta-schardinger dextrin-as adsorbent, with reference to micro matrix solid phase dispersion abstraction technique, for extracting Flos Lonicerae in
The new method of phenolic acid effective constituents.
Background technology
Flos Lonicerae just opens for the dry flower or band of caprifoliaceae plant Radix Ophiopogoniss (Lonicera japonica Thunb.)
Flower.Flos Lonicerae has heat-clearing and toxic substances removing, the effect of dispelling wind and heat pathogens.For carbuncle swells furunculosis, sore throat, erysipelas, toxic-heat and blood stasis, wind heat sense
Emit, warm disease heating etc..Chlorogenic acid (chlorogenic acid), neochlorogenic acid (neochlorogenic acid), isochlorogenic acid
(isochlorogenic acid) is the main active of Flos Lonicerae.Isochlorogenic acid is a mixture, and 3,5- bis- caffeoyls
Quininic acid (3,5-O-dicaffeoylquinic acid) and Isochlorogenic acid C (4,5-O-
Dicaffeoylquinic acid) it is its main component.It is reported that, chlorogenic acid compound has antibacterial, function of gallbladder promoting, hemostasis, increasing
Plus the pharmacological action such as leukocyte.And there is the pharmacological action for significantly increasing gastrointestinal peristalsiss and promoting gastric secretion.Wherein content is most
It is high for chlorogenic acid, next to that 3,5-diCQA, Isochlorogenic acid C and neochlorogenic acid.
Traditional Traditional Chinese medicine extraction method has infusion process, THROUGH METHOD, circumfluence method etc., and they generally have consumption of organic solvent
Greatly, extraction time is longer, operating process is loaded down with trivial details, the low shortcoming of extraction ratio.And these methods easily lose effective ingredient, produce
Larger error, the use of a large amount of organic solvents affects larger to healthy, and has larger pollution to environment, does not meet green
The proposal of colour chemistry.Solid-Phase Extraction (Solid-Phase Extraction, SPE) is a kind of specimen preprocessing that developed recently gets up
Reason technology, is mainly used in separation, purification and the concentration of sample, and comparing traditional method can improve the response rate of analyte, make
Analyte is efficiently separated with interfering component, reduces sample pretreatment process, simple to operate quick.It is widely used in doctor
The fields such as medicine, environment, food, commodity inspection.But it also has, and consumption of organic solvent is big, poor repeatability, easily blocking, flow is low, sectional area
Little the shortcomings of.
In view of above-mentioned limitation, many new extracting method have developed on the basis of Solid-Phase Extraction, mainly including base
The extraction of matter solid phase dispersion, molecular engram solid phase extraction, dispersive liquid-liquid microextraction, Magnetic solid phases extraction etc..Compare traditional solid phase
This kind of new extracting process of extracting process has more significant advantage, mainly has that consumption of organic solvent is few, environmental pollution is little, behaviour
Make simple and quick, extraction efficiency high.Wherein matrix solid phase dispersion abstraction technique just has much representativeness.
Dispersive solid phase extraction (MSPD, matrix solid-phase dispersion) is U.S. Louisiana states
The Barker of vertical university teaches a kind of quick sample treatment technology set up in 1989.Its principle is by solid-phase adsorbent and sample
Product grind together, obtain the mixture of leather hard and fill post as filler, then with different eluent solvent pillars, will
Various determinands are eluted.It is by the homogenizing of sample, extraction, concentration and purifies a step and completes, it is to avoid sample in homogenizing, carry
The loss of the measured object caused during taking, purify etc., while the simplicity for becoming the pretreatment of sample, improves analyze speed,
Reduce the consumption of organic solvent, the extracting solution for obtaining mostly can direct analysis detection, reduce operating procedure and to environment
Pollution.In document report currently for the matrix solid phase dispersion abstraction technique of natural product, its medical material, adsorbent and eluting
The consumption of agent is all larger, not only waste of materials but also very big pollution has been caused to environment.
One committed step of matrix solid phase dispersion extraction is the selection of adsorbent, is presently used for matrix solid phase dispersion extraction
The adsorbent for taking mainly includes silica gel, C18, activated carbon, aluminium oxide, florisil silica etc..
1891, Villier made to isolate ring paste in used potato starch first from Bacillus amylobacter
Essence.Cyclodextrin (Cyclodextyin, CD) is to act on starch, glycogen, Fructus Hordei Germinatus oligomerization by cyclodextrin glucose residue transferring enzyme
The glucose polymers such as sugar and formed, the ring being formed by connecting with α-Isosorbide-5-Nitrae-glucoside bond by 6-12 D- glucopyranosyl
Shape oligosaccharide.Common mainly has tri- kinds of cyclodextrin α, β, γ.Wherein beta-schardinger dextrin-is cheap and easy to get, most widely used, and it has " interior
Chamber is hydrophobic, and outer wall is hydrophilic " special construction and property, and be in " truncated cones " shape in external form, therefore can be in water phase middle mold
Intend the hydrophobic pocket of enzyme, with a series of organic molecules Inclusion Complexes are formed, be widely used in absorption and the extraction of environmental contaminants
Take, the design of the imitative enzyme system of the inclusion of water-insoluble drug molecule and solubilising, supermolecule with the field such as construct.And beta-schardinger dextrin-
Cavity size is moderate, and Binding ability is strong, can be absorbed in human body, is decomposed, safe to the human body nontoxic, therefore suitable as substrate
The adsorbent of solid phase dispersion extraction.
Up to now, matrix solid phase dispersion abstraction technique is applied to as adsorbent using beta-schardinger dextrin-, in the field of extraction still
Report is had no, the method is blank for the extraction detection of Flos Lonicerae is even more a piece of research.
The content of the invention
The present invention is to solve there is few adsorbent selectivity, medical material and adsorbent in existing matrix solid phase dispersion abstraction technique
Consumption is big, the problem that organic solvent consumption is big, and provide by the use of beta-schardinger dextrin-as adsorbent, micro matrix solid phase dispersion
The method of effective ingredient in extraction Chinese medicine honeysuckle.Beta-schardinger dextrin-is used as the target molecule absorption in adsorbent and Chinese medicine honeysuckle
With reference to, target molecule rapid extraction, eluting from medicinal powder, and the present invention is implemented in a micro system, compare medicine
The extracting method of allusion quotation, greatly reduces the usage amount of organic solvent.
The technical solution used in the present invention is：
A kind of method of phenolic acid effective constituents in extraction Chinese medicine honeysuckle, the phenolic acid effective constituents are that fresh green is former
One or more in acid, chlorogenic acid, 3,5-diCQA, Isochlorogenic acid C, methods described bag
Include following steps：
Flos Lonicerae sample is weighed, in being placed in mortar, cyclodextrin solid phase extraction filler is added, is sufficiently mixed with medical material sample and is ground
Mill.Solid phase extraction column is separately taken, bottom adds sieve plate, with ground powder filling pillar, top adds again after filler is tamped
Enter sieve plate, the pillar filled in is placed on solid-phase extracting instrument, eluting is carried out to pillar, eluent is collected in bottom with centrifuge tube, from
The heart, takes the supernatant and is phenolic acids extracts active ingredients liquid, is tested and analyzed with Ultra Performance Liquid Chromatography instrument.
The species of the cyclodextrin is：Alpha-cyclodextrin, beta-schardinger dextrin-, gamma-cyclodextrin, methyl-B-cyclodextrin, two hydroxypropyls
Group-beta-cyclodextrin, preferably beta-schardinger dextrin-.
The Honeysuckle Flower is 1: 0.25～4, preferably 1: 3. with the mass ratio of beta-schardinger dextrin-adsorbent
The milling time is 60～160s, preferably 120s.
The species of the eluant has：Methanol, 80% methanol-water, 90% methanol-water, acetonitrile, ethanol, methanol：Ethanol 1: 1,
Preferably 80% methanol-water.
It is preferred that after specific experiment operating procedure be：
Weigh Chinese medicine honeysuckle powder 25mg, beta-schardinger dextrin -75mg is weighed according to mass ratio 1: 3, add in mortar and grind
120s, takes 1mL specification solid phase extraction columns, and bottom adds sieve plate, and with ground powder filling pillar, top adds again after tamping
Enter sieve plate, the pillar filled in is placed on solid-phase extracting instrument, take 0.5mL80% methanol-waters, eluting is carried out to pillar, bottom is used
1.5mL specifications centrifuge tube collect eluent, after be put in centrifuge, 13000rpm centrifugation 5min, take the supernatant and be phenolic acid
Effective constituents extracting solution, loads sample injection bottle, with Ultra Performance Liquid Chromatography instrument (UPLC) sampling analyses, obtains the liquid phase of extracting solution
Chromatogram；
Then a series of reference substance solution of variable concentrations is prepared with the reference substance of neochlorogenic acid, by Flos Lonicerae extractive solution
Similarity condition detects that the liquid chromatogram of acquisition neochlorogenic acid reference substance is compareed with neochlorogenic acid with Ultra Performance Liquid Chromatography instrument
The concentration of product is abscissa, bent as the standard that vertical coordinate draws neochlorogenic acid with the peak area of neochlorogenic acid reference substance chromatographic peak
Line, makes in the same way chlorogenic acid standard curve, 3,5-diCQA standard curve, Isochlorogenic acid C
Standard curve；According to the standard curve of the peak area of each chromatographic peak and each composition in the liquid chromatogram of extracting solution, it is calculated
The content of neochlorogenic acid, chlorogenic acid, 3,5-diCQA, Isochlorogenic acid C in extracting solution, can accordingly change
Calculation obtains the content of the effective ingredient relative to Chinese medicine honeysuckle powder of extraction in extracting solution.
It is an advantage of the current invention that：
The adsorbent that this method is extracted as matrix solid phase dispersion first using beta-schardinger dextrin-to Chinese crude drug extracting
Detection, with originality, is not reported in current documents and materials.
This method is that extraction detection is carried out under micro system, compares the extracting method described in Chinese Pharmacopoeia and significantly reduces
The use of organic solvent, reduces the pollution to environment.Additionally, the usage amount of medical material, adsorbent is also extremely low, resource has been saved
Also improve environmental protection benefit simultaneously.
Special construction of the adsorbent cyclodextrin of the present invention with " inner chamber is hydrophobic, and outer wall is hydrophilic " and property, and
It is in " truncated cones " shape in external form, therefore sorption extraction efficiency high and beta-schardinger dextrin-cavity size is moderate, Binding ability is strong,
Can be absorbed in human body, decomposed, it is safe to the human body nontoxic, it is free from environmental pollution and cheap and easy to get.So that experimental implementation environment peace
It is complete reliable, in the today for advocating Green Chemistry, meet sustainable development and environmental friendliness theory.
The inventive method possesses a series of characteristic of excellent protrusions：It is nontoxic to human body, free from environmental pollution；Operation environment safety
It is reliable；For the extraction of natural drug, the present invention also has the advantages that efficiency high, operating procedure rapid and convenient.
Description of the drawings
Fig. 1 is the process chart of matrix solid phase dispersion extracting process of the present invention.
Fig. 2 is the matrix solid phase dispersion effect of extracting block diagram for investigating adsorbent species.In figure, 1,2,3,4 represent gold silver
Spend middle different effective ingredient, 1：Neochlorogenic acid, 2：Chlorogenic acid, 3：3,5-diCQA, 4：4,5- bis- caffeoyl Kuis
Thujic acid.
Fig. 3 is to investigate the matrix solid phase dispersion effect of extracting column between medical material and beta-schardinger dextrin-under different quality ratio
Figure.In figure, 1,2,3,4 represent respectively effective ingredient different in Flos Lonicerae, and 1：Neochlorogenic acid, 2：Chlorogenic acid, 3：3,5- bis- coffees
Coffee acyl quininic acid, 4：Isochlorogenic acid C.Abscissa represents the quality of beta-schardinger dextrin-, unit mg in figure.
Fig. 4 is the matrix solid phase dispersion effect of extracting block diagram for investigating eluant species.In figure, 1,2,3,4 represent respectively
Different effective ingredient in Flos Lonicerae, 1：Neochlorogenic acid, 2：Chlorogenic acid, 3：3,5-diCQA, 4：4,5- bis- coffees
Acyl quininic acid.
Fig. 5 is the matrix solid phase dispersion effect of extracting broken line graph for investigating different milling times.In figure, 1,2,3,4 generations respectively
Different effective ingredient in table Flos Lonicerae, 1：Neochlorogenic acid, 2：Chlorogenic acid, 3：3,5-diCQA, 4：4,5- bis- coffees
Coffee acyl quininic acid.Abscissa represents milling time in figure.
Fig. 6 is the liquid chromatogram that Flos Lonicerae mixes reference substance.In figure, 1,2,3,4 represent respectively it is different in Flos Lonicerae
Effective ingredient, 1：Neochlorogenic acid, 2：Chlorogenic acid, 3：3,5-diCQA, 4：Isochlorogenic acid C.
Fig. 7 is the liquid chromatogram of Flos Lonicerae phenolic acids extracts active ingredients liquid.In figure, 1,2,3,4 represent in Flos Lonicerae
Effective ingredient, be followed successively by：1：Neochlorogenic acid, 2：Chlorogenic acid, 3：3,5-diCQA, 4：4,5- bis- caffeoyl quinines
Acid.
Specific embodiment
Detection method provided by the present invention is made a more detailed description by following instance.Because it applies model
Enclose wide, therefore specific embodiment is also more, the discussion below in conjunction with several examples is further elaborated to present disclosure.
The preparation method of Flos Lonicerae reference substance solution is concretely comprised the following steps：Take neochlorogenic acid, chlorogenic acid, the caffeoyl Kuis of 3,5- bis-
Thujic acid, Isochlorogenic acid C reference substance it is appropriate, accurately weighed, in putting brown measuring bottle, plus methanol makes every 1mL containing new
The μ g of chlorogenic acid 500, the μ g of chlorogenic acid 500, the μ g of 3,5-diCQA 500, the control of the μ g of Isochlorogenic acid C 500
Product solution, obtains final product.
Embodiment 1
Weigh each 25mg of 5 parts of Chinese medicine honeysuckle powder to be separately added in 5 groups of mortars, and weigh different types of cyclodextrin
(alpha-cyclodextrin, beta-schardinger dextrin-, gamma-cyclodextrin, methyl-B-cyclodextrin, dihydroxypropyl-beta-schardinger dextrin -) each 25mg, adds 5 groups and grinds
With Chinese medicine honeysuckle powder mull 120s in alms bowl.5 1mL specification solid phase extraction columns are taken, bottom adds sieve plate, respectively with grinding
The powder filling pillar of milled, top adds sieve plate after tamping, and is placed on solid-phase extracting instrument.0.5mL methanol is taken, pillar is injected
It is interior, eluting is carried out one by one to pillar.Bottom is collected respectively with 5 1.5mL specifications centrifuge tubes, after be put in centrifuge,
13000rpm is centrifuged 5min.The supernatant is taken, phenolic acids extracts active ingredients liquid as in Flos Lonicerae is separately sampled to use ultra high efficiency
Chromatograph of liquid (UPLC) sampling analyses.
Embodiment instrument be Agilent Ultra Performance Liquid Chromatography system (the Infinity LC of Agilent 1290,
Agilent Technologies, Santa Clara, CA, USA) it is equipped with vacuum air pump, binary pump flow phase system, constant temperature
Automatic sampler, thermostatted column compartment.Chromatographic column：Agilent Zorbax Extend-C18 column (2.1mm × 50mm, 1.8
μm), Detection wavelength：330nm.Column temperature：40℃.Sample size：1μL.Flow velocity 0.4mL/min, mobile phase：A：0.1v% formic acid waters,
B：Methanol.Gradient elution：0～2min, 0%～30%B；2～3min, 30%～35%B；3～4min, 35%～40%B；4
～7min, 40%～50%B；7～8min, 50%～100%B；8～9min, 100%～100%B；9～10min, 100%
～30%B, equilibration time 5min.
Experimental result such as table 1 below, the data in table 1 are peak area.
Table 1.
1 represents neochlorogenic acid, 2 represents chlorogenic acid, 3 represent 3,5-diCQA, 4 represent the caffeoyl Kuis of 4,5- bis-
Thujic acid.
The Solid-Phase Extraction effect block diagram of variety classes adsorbent is as shown in Figure 2.As a result show, the extraction effect of beta-schardinger dextrin-
Fruit is optimal.The solution that beta-schardinger dextrin-is eluted is yellow green and most deep.Because of the molecule hole of beta-schardinger dextrin-it is moderate so that
It can preferably adsorb the active component in Flos Lonicerae.
Embodiment 2
Weigh each 25mg of 9 parts of Chinese medicine honeysuckle powder to be separately added in 9 groups of mortars, and weigh different quality (6.25mg,
12.5mg, 25mg, 37.5mg, 50mg, 62.5mg, 75mg, 87.5mg, 100mg) beta-schardinger dextrin-, in being separately added into 9 groups of mortars
With Chinese medicine honeysuckle powder mull 120s.9 1mL specification solid phase extraction columns are taken, bottom adds sieve plate, respectively with ground
Powder filling pillar, tamp after top add sieve plate.Fill in 9 pillars are placed on solid-phase extracting instrument.Take 0.5mL first
Alcohol, to pillar eluting is carried out one by one.Bottom is collected respectively with 9 1.5mL specifications centrifuge tubes, after be put in centrifuge,
13000rpm is centrifuged 5min.Supernatant dress sample is taken, with Ultra Performance Liquid Chromatography instrument (UPLC) sampling analyses.
Experimental result such as table 2 below, the data in table 2 are peak area.
Table 2.
1 represents neochlorogenic acid, 2 represents chlorogenic acid, 3 represent 3,5-diCQA, 4 represent the caffeoyl Kuis of 4,5- bis-
Thujic acid.
Mass ratio between different medical material and beta-schardinger dextrin-, the such as Fig. 3 of the impact to matrix solid phase dispersion effect of extracting institutes
Show.As a result show, as the increase effect of extracting of beta-schardinger dextrin-consumption strengthens successively, but increase to after 75mg, effect of extracting tends to
It is gentle even to have declined, so from 75mg being optimal adsorbent amount in follow-up experiment.Possible the reason for is, with
The increase of adsorbent amount, can preferably adsorb the active component in hybrid solid, but the adsorbable total amount of active component
Limited, adsorbent amount is increased to after optimal value, and effect of extracting tends to gentle, illustrates that active component has been adsorbed agent substantially
Adsorbed.And excessive adsorbent also results in active component desorbing from adsorbent and becomes difficult, and extraction effect is caused on the contrary
Fruit has downward trend.
Embodiment 3
Weigh each 25mg of 6 parts of dried orange peel medicinal powders to be separately added in 6 groups of mortars, and weigh 6 parts of 75mg beta-schardinger dextrin -s, respectively
Add the times (60s, 80s, 100s, 120s, 140s, 160s) different from Chinese medicine honeysuckle powder mull in 6 groups of mortars.Take 6
The solid phase extraction column of 1ml specifications is propped up, bottom adds sieve plate, respectively with ground powder filling pillar, top after tamping
Add sieve plate.Fill in 6 pillars are placed on solid-phase extracting instrument.Take the methanol of 0.5ml, in injection pillar, to pillar by
One carries out eluting.Bottom is collected respectively with the centrifuge tube of 6 1.5ml specifications, in being put into ion instrument, 13000rpm centrifugation 5min.
Supernatant dress sample is taken, with Ultra Performance Liquid Chromatography instrument (UPLC) sampling analyses.
Experimental result such as table 3 below, the data in table 3 are peak area.
Table 3.
1 represents neochlorogenic acid, 2 represents chlorogenic acid, 3 represent 3,5-diCQA, 4 represent the caffeoyl Kuis of 4,5- bis-
Thujic acid.
The matrix solid phase dispersion effect of extracting block diagram of different milling times is shown in Fig. 4.As a result show, with milling time
Increase, effect of extracting becomes larger, and to optimal value 120s, with the increase of milling time, effect of extracting has on the contrary decline
Trend.Possible the reason for is that sufficiently grinding will be helpful to the active component that adsorbent is more fully contacted in hybrid solid, from
And increase effect of extracting.But the grinding of excess can cause active component to be forced to be squeezed in less in adsorbent, fine and close aperture
In so that eluting becomes difficult, and effect of extracting is reduced on the contrary.
Embodiment 4
Weigh each 25mg of 6 parts of Chinese medicine honeysuckle powder to be separately added in 6 groups of mortars, and weigh 6 parts of 75mg beta-schardinger dextrin -s, point
Do not add in 6 groups of mortars with Chinese medicine honeysuckle powder mull 120s.6 1mL specification solid phase extraction columns are taken, bottom adds sieve
Plate, respectively with ground powder filling pillar, top adds sieve plate after tamping.Fill in 6 pillars are placed in into solid phase extraction
Take on instrument.Take the different types of eluant of 0.5mL (methanol, 80% methanol, 90% methanol, ethanol, acetonitrile, methanol：Ethanol is 1:
1), it is injected separately in pillar, eluting is carried out one by one to pillar.Collected with 6 1.5mL specifications centrifuge tubes respectively bottom.Eluting is tied
Shu Hou, in being put into centrifuge, 13000rpm centrifugation 5min.Supernatant dress sample is taken, is adopted with Ultra Performance Liquid Chromatography instrument (UPLC)
Sample is analyzed.
Experimental result such as table 4 below, the data in table 4 are peak area.
Table 4.
1 represents neochlorogenic acid, 2 represents chlorogenic acid, 3 represent 3,5-diCQA, 4 represent the caffeoyl Kuis of 4,5- bis-
Thujic acid.
The matrix solid phase dispersion effect of extracting block diagram of variety classes eluant is shown in Fig. 5.As a result show, methanol-eluted fractions effect
Good compared with other eluent effect of extracting, possible methanol can mutually produce hydrogen bond action, by contrast, second and target molecule between
The experimental result that one group of nitrile is worst in all study groups one group, is solvability of the acetonitrile to target molecule the reason for possible
And interact with adsorbent most weak.And 80% methanol extraction best results are likely due to polarity and most connect with target molecule
Closely.
Withinday precision
Chinese medicine honeysuckle powder 25mg, beta-schardinger dextrin -75mg are weighed, is added in mortar and is ground 120s.Take 1mL specification solid phases
Extraction pillar, bottom adds sieve plate, and with ground powder filling pillar, top adds sieve plate after tamping.It is little by what is filled in
Post is placed on solid-phase extracting instrument.0.5mL80% methanol-waters are taken, in injection pillar, eluting is carried out to pillar.Advised with 1.5mL bottom
Lattice centrifuge tube is collected.After eluting terminates, in being put into centrifuge, 13000rpm centrifugation 5min.Supernatant dress sample is taken, ultra high efficiency is used
Chromatograph of liquid (UPLC) sampling analyses.In interior continuous sample introduction 6 times on the same day.
Day to day precision
Chinese medicine honeysuckle powder 25mg, beta-schardinger dextrin -75mg are weighed, is added in mortar and is ground 120s.Take 1mL specification solid phases
Extraction pillar, bottom adds sieve plate, and with ground powder filling pillar, top adds sieve plate after tamping.It is little by what is filled in
Post is placed on solid-phase extracting instrument.0.5mL80% methanol-waters are taken, in injection pillar, eluting is carried out to pillar.Advised with 1.5mL bottom
Lattice centrifuge tube is collected.After eluting terminates, in being put into centrifuge, 13000rpm centrifugation 5min.Supernatant dress sample is taken, ultra high efficiency is used
Chromatograph of liquid (UPLC) sampling analyses.By the sample continuous sample introduction 3 days, daily 2 times.
In a few days, day to day precision experimental result is summarized as follows table 5：
Table 5
1 represents neochlorogenic acid, 2 represents chlorogenic acid, 3 represent 3,5-diCQA, 4 represent the caffeoyl Kuis of 4,5- bis-
Thujic acid.
Repeatability is investigated
It is parallel to do 3 groups with reference to following experiments step, as repeatability investigation.
Chinese medicine honeysuckle powder 25mg, beta-schardinger dextrin -75mg are weighed, is added in mortar and is ground 120s.Take 1mL specification solid phases
Extraction pillar, bottom adds sieve plate, and with ground powder filling pillar, top adds sieve plate after tamping.It is little by what is filled in
Post is placed on solid-phase extracting instrument.0.5mL80% methanol-waters are taken, in injection pillar, eluting is carried out to pillar.Advised with 1.5mL bottom
Lattice centrifuge tube is collected.After eluting terminates, in being put into centrifuge, 13000rpm centrifugation 5min.Supernatant dress sample is taken, ultra high efficiency is used
Chromatograph of liquid (UPLC) sampling analyses.
Medical material assay
Chinese medicine honeysuckle powder 25mg, beta-schardinger dextrin -75mg are weighed, is added in mortar and is ground 120s.Take 1mL specification solid phases
Extraction pillar, bottom adds sieve plate, and with ground powder filling pillar, top adds sieve plate after tamping.It is little by what is filled in
Post is placed on solid-phase extracting instrument.0.5mL80% methanol-waters are taken, in injection pillar, eluting is carried out to pillar.Advised with 1.5mL bottom
Lattice centrifuge tube is collected.After eluting terminates, in being put into centrifuge, 13000rpm centrifugation 5min.The supernatant is taken, as phenolic acids have
Effect constituents extraction liquid, with Ultra Performance Liquid Chromatography instrument (UPLC) sampling analyses.Obtain the liquid chromatogram of extracting solution.
Fig. 7 is the liquid chromatogram of phenolic acids extracts active ingredients liquid.In figure, 1,2,3,4 represent respectively in Flos Lonicerae not
Same effective ingredient, respectively：1：Neochlorogenic acid, 2：Chlorogenic acid, 3：3,5-diCQA, 4：4,5- bis- caffeoyl Kuis
Thujic acid.
Prepared not with the reference substance of neochlorogenic acid, chlorogenic acid, 3,5-diCQA, Isochlorogenic acid C
With the mixed reference substance solution of concentration, detected with Ultra Performance Liquid Chromatography by similarity condition, the chromatogram of acquisition reference substance,
As abscissa, the peak area of chromatographic peak is vertical seat to sample size with four reference substances with the chromatogram of four reference substance solution
Mark, makes respectively neochlorogenic acid standard curve, chlorogenic acid standard curve, 3,5-diCQA standard curve and 4,5- bis-
Caffeoylquinic acids standard curve.Fresh green in Chinese medicine honeysuckle is calculated according to the liquid chromatogram and standard curve of extracting solution
Ortho acid, chlorogenic acid, 3,5-diCQA, the content of Isochlorogenic acid C.
Neochlorogenic acid, chlorogenic acid, 3,5-diCQA, Isochlorogenic acid C concentration are 100 μ g/mL
The liquid chromatogram of Flos Lonicerae reference substance solution represent different in Flos Lonicerae have respectively as shown in fig. 6, in figure, 1,2,3,4
Effect composition, respectively：1：Neochlorogenic acid, 2：Chlorogenic acid, 3：3,5-diCQA, 4：Isochlorogenic acid C.
The standard curve and test limit of 4 kinds of compositions and quantitative limit are as shown in table 6 below：
Table 6.
The response rate is tested
Chinese medicine honeysuckle powder 25mg, beta-schardinger dextrin -75mg are weighed, parallel to do 2 groups, one of which adds 100 μ μ g/mL's
Flos Lonicerae mixes the μ L of reference substance 50, and another group is not added with Flos Lonicerae mixing reference substance, and two groups add in mortar and grind 120s.Respectively
2 1mL specification solid phase extraction columns are taken, bottom adds sieve plate, with ground powder filling pillar, top adds after tamping
Sieve plate.The pillar filled in is placed on solid-phase extracting instrument.0.5mL80% methanol-waters are taken, is injected separately in pillar, pillar is carried out
Eluting one by one.Collected with 1.5mL specifications centrifuge tube bottom.After eluting terminates, in being put into centrifuge, 13000rpm centrifugations
5min.Supernatant dress sample is taken, with Ultra Performance Liquid Chromatography instrument (UPLC) sampling analyses.
Repeatability, assay, response rate experimental result are summarized as follows table 7：
Table 7
As a result show, the repeatability of the inventive method is good, and the response rate is high, and detection accuracy is high.
Reference examples：
It is according to the extracting method in Chinese Pharmacopoeia：This product powder (crossing No. four sieves) about 0.5g, it is accurately weighed, put tool plug cone
In shape bottle, precision adds the methanol 50ml of people 50%, weighed weight, supersound process (power 250W, frequency 35kHz) 30 minutes to let cool,
Again weighed weight, with 50% methanol the weight of less loss is supplied, and is shaken up, and filtration, precision measures subsequent filtrate 5ml, puts 25ml brown amounts
In bottle, plus 50% methanol shakes up to scale, obtains final product.
Gained extracting solution Ultra Performance Liquid Chromatography instrument (UPLC) sampling analyses, obtain liquid chromatogram and standard curve pair
According to it is neochlorogenic acid 2.692mg/g, chlorogenic acid relative to the content of Chinese medicine honeysuckle that can measure effective ingredient in extracting solution
25.05mg/g, 3,5-diCQA 16.918mg/g, Isochlorogenic acid C 2.3375mg/g.And the application
Effective ingredient is neochlorogenic acid 3.3719mg/g, chlorogenic acid relative to the content of Chinese medicine honeysuckle in extracting solution obtained in method
33.3368mg/g, 3,5-diCQA 20.733mg/g, Isochlorogenic acid C 2.7641mg/g, compare medicine
The method extraction effect of allusion quotation is significantly improved.
Claims (6)
1. a kind of Trace bio-element method of natural product, for extracting Chinese medicine honeysuckle in liposoluble ingredient, the phenolic acids
Effective ingredient is one or two in neochlorogenic acid, chlorogenic acid, 3,5-diCQA, Isochlorogenic acid C
More than, the method comprising the steps of：
(1) Chinese medicine honeysuckle powder and adsorbent are weighed with the mass ratio of certain medical material and adsorbent, adds in mortar and grind
Regular hour.1mL specification solid phase extraction columns are taken, bottom adds sieve plate, with ground solid-filling pillar, filler filled out
Top adds sieve plate after reality.The pillar filled in is placed on solid-phase extracting instrument.The eluent of appropriate volume is taken, pillar is injected
It is interior, the air pump of solid-phase extracting instrument is opened, eluting is carried out to pillar.Collected with 1.5mL specifications centrifuge tube bottom.Eluting terminates
Afterwards, by liquid vortex 10s in centrifuge tube, after be put in centrifuge be centrifuged.Supernatant dress sample is taken, Ultra Performance Liquid Chromatography instrument is used
(UPLC) sampling analyses, obtain the liquid chromatogram of phenolic acid effective constituents in extracting solution；
(2) reference substance solution of variable concentrations is prepared with the reference substance of neochlorogenic acid, according to extracting solution similarity condition ultra high efficiency
Chromatograph of liquid detection, obtains the liquid chromatogram of neochlorogenic acid reference substance, with the sample size of neochlorogenic acid reference substance as horizontal seat
Mark, the peak area of chromatographic peak makes neochlorogenic acid standard song as vertical coordinate with the liquid chromatogram of neochlorogenic acid reference substance solution
Line, makes in the same way chlorogenic acid standard curve, 3,5-diCQA standard curve, Isochlorogenic acid C
Standard curve；According to the peak area and the standard curve of each composition of chromatographic peak in the liquid chromatogram of each composition of extracting solution, meter
Calculation obtains the content of neochlorogenic acid in extracting solution, chlorogenic acid, 3,5-diCQA, Isochlorogenic acid C.
2. the method for claim 1, it is characterised in that in the step (1), the adsorbent is beta-schardinger dextrin-.
3. the method for claim 1, it is characterised in that in the step (1), certain medical material and adsorbent
Mass ratio is 1: 3.
4. the method for claim 1, it is characterised in that in the step (1), milling time is 120s.
5. the method for claim 1, it is characterised in that in the step (1), the eluent is 80% methanol.
6. the method for claim 1, it is characterised in that in the step (1), the volume of the eluent is 0.5mL.
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| CN113702549A (en) * | 2021-07-15 | 2021-11-26 | 杭州师范大学 | Miniature extraction method of lotus plumule |
| CN115343390A (en) * | 2022-08-22 | 2022-11-15 | 杭州师范大学 | Method for micro-extracting effective and harmful components in traditional Chinese medicine by matrix solid phase dispersion |
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| CN113702549A (en) * | 2021-07-15 | 2021-11-26 | 杭州师范大学 | Miniature extraction method of lotus plumule |
| CN115343390A (en) * | 2022-08-22 | 2022-11-15 | 杭州师范大学 | Method for micro-extracting effective and harmful components in traditional Chinese medicine by matrix solid phase dispersion |
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