CN1219214C - Novel process for determining herb character - Google Patents
Novel process for determining herb character Download PDFInfo
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- CN1219214C CN1219214C CN 02124697 CN02124697A CN1219214C CN 1219214 C CN1219214 C CN 1219214C CN 02124697 CN02124697 CN 02124697 CN 02124697 A CN02124697 A CN 02124697A CN 1219214 C CN1219214 C CN 1219214C
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Abstract
The present invention relates to a novel method for fast separating and identifying active ingredients of herb medicine. The separating method which does not need complex and time-consuming chromatography or HPLC operation is effective. On the other hand, the present invention relates to a novel method for determining herb medicine quality. According to the bioactive identified results of the herb medicine quality, the treated plastic slide glass is applied to the quality control of herb medicine constituents.
Description
Technical field
The present invention relates to the novel method of a kind of quick separation and Rapid identification herbal medicine active ingredient.Separation method is effectively and need not loaded down with trivial details and time-consuming chromatography or HPLC work.Another aspect of the present invention relates to a kind of novel method of definite herb character, and it is according to its bioactive qualification result, and treated plastic slide is applied to the quality control of herbal ingredients.
Background of invention
For many years, herbal medicine (whole strain plant) is applied in the medical treatment.Normally take herbal medicine, or cover herbal medicine with the mode external application paste of paste in the mode of infusion liquid or tea.Yet practical experience shows, when handling in an identical manner, still there is significant otherness on the medical functions in the individuality of the herbal medicine of same breed.
The composition of herbal medicine is the potpourri of chemical compound lot normally, and wherein some compounds may biologically active and may be had therapeutic efficiency to human body and animal.Classes of compounds and/or its relative content may change in the composition of herbal medicine, natural condition when the hereditary information of herbal medicine individuality and the growth of this herbal medicine are depended in this change, geographic position such as plantation, soil constitution, water quality, weather (comprising temperature and humidity), solar radiation intensity and growth time.
Herbal medicine depends on several reactive compounds and the relative content thereof that this herbal medicine contains to the medical functions of human body and animal.The reactive compound amount that the herbal medicine individuality is contained is higher, the individual high therapeutic efficiency of healing that shows of this herbal medicine.Prior art there is no guide to detecting required active ingredient and the relative content thereof that herbal medicine contained, even also is like this when this herbal medicine is known when having specific therapeutic efficiency.Judge known human body and animal to be had the quality of the herbal medicine individuality of therapeutic efficiency, usually based on the experience of being familiar with the herbal medicine personage, it is build, outward appearance, the color of herbal medicine according to the observation, the tissue fluid of smelling the smell of news herbal medicine and/or chewing herbal medicine.Human body and animal are had the herbal medicine of therapeutic efficiency, and prior art not teaching or hint can be determined the quality (that is, having required active ingredient and/or its relative content) of this herbal medicine by scientific methods.
U.S. Patent number 6,156, but the method for the pharmacologically active potpourri of the chemical analysis of a kind of re-extract plant of 291 announcements, wherein this method can be improved the quality control of the potpourri of this pharmaceutically-active ingredients.
For addressing this problem, discovery according to the inventor, the invention provides a kind of effectively, science and detect and determine to be present in the method for the active ingredient in the herbal medicine individuality fast, filter out herbal medicine individuality with active ingredient and required therapeutic efficiency with this method.
The invention summary
The invention provides the novel method of a kind of quick separation and Rapid identification herbal medicine active ingredient, it comprises the following step:
(a) obtain the herbal medicine extract with the solvent extraction herbal medicine, then this herbal medicine extract of evaporation and concentration;
(b) the concentrated herbal medicine extract of order mixes mutually with water and water-miscible organic solvent, removes formed insoluble particle then to obtain component A;
(c) component A is mixed with water and water-insoluble organic solvents and component A is distributed extraction, and layering, organic component B and water component E obtained;
(d) mixed water and said components B and distribute extraction obtain organic component C and water component D;
(e) mix water-insoluble organic solvents and said components E and distribute extraction, obtain organic component F and water component G;
(f) the component A-G with gained is added to respectively on the plastic slide of having anticipated; With
(g) carry out hybridization reaction and input to detect the active ingredient that is present in the herbal medicine extracted component.
The present invention relates to a kind of novel method of definite herb character on the other hand, and it is according to its bioactive qualification result, and treated plastic slide is applied to the quality control of herbal ingredients.
Another aspect of the present invention provides a kind of novel method of definite herb character, and it is to be present in the biological activity that can combine with tumor necrosis factor-alpha (TNF-α) selectivity in the herbal medicine by the technology of utilizing biochip to detect.
The accompanying drawing summary
Fig. 1 has shown the fluoroscopic image of the resulting extracted component A-G of the present invention.
Fig. 2 has shown the standard fabrication step of herbal medicine extracted component.
Detailed Description Of The Invention
The invention provides the novel method of a kind of quick separation and Rapid identification herbal medicine active ingredient.The included separating step of method of the present invention is effectively, and compares with chromatography or HPLC, need not to carry out loaded down with trivial details and time-consuming job.The step of the distribution extraction of herbal medicine of the present invention is specified in Fig. 2.
In step (a), earlier herbal medicine is ground to form attritive powder, then obtain the herbal medicine extract with solvent extraction.In the step (a) used solvent comprise water, alkanols, ethers, and composition thereof.Solvent preferably has the alkanol of 1-6 carbon atom, special particular methanol.The ratio of herbal medicine and solvent is about 1: 30 to about 1: 50 (w/v), preferred about 1: 40 (w/v).Then merge herbal medicine extract and evaporation and concentration and obtain a potpourri.
In the step (b), make in the step (a) resulting concentrated carbinol mixture again with water and water-miscible organic solvent with about 2-4: 0.5-1.5: 0.5-1.5 (v: v: v), preferably about 3: 1: 1 (v: v: violent mixing of ratio v) and extracting.Centrifugal to remove the supernatant that can clarify behind insoluble particle/agglomerate, be component A.Used water-miscible organic solvent preferably has the alkanol of 1-6 carbon atom in the step (b), special preferred alcohol.
In the step (c), component A distributes between water and water-insoluble organic solvents and then separates, and obtains organic component B and water component E.Component A: water: the ratio of water-insoluble organic solvents be about 0.5-1.5: 0.5-1.5: 0.5-1.5 (v: v: v), preferably about 1: 1: 1 (v: v: v).Used water-insoluble organic solvents is the carboxylate of a tool 3-10 carbon atom, preferably ethyl acetate in the step (c).
Step (d) and (e) in, organic component B and water component E further distribute between water and water-insoluble organic solvents respectively and then separate, through this step, organic component B obtains organic component C and water component D, and water component E obtains organic component F and water component G.Step (d) and (e) in distribution be that ratio in water and water-insoluble organic solvents is about 0.5-1.5: 0.5-1.5 (v/v), carry out under the condition of preferred about 1: 1 (v/v).Step (d) and (e) in used organic solvent ethyl acetate preferably.
The component A-G of gained is added to respectively on the coating plastic slide glass of having anticipated, and then under the known condition of those skilled in the art, carries out hybridization reaction and input.
Above-mentioned each herbal ingredients can contain a kind of herbal medicine extracted component or the potpourri of herbal medicine extracted component.Above-mentioned herbal medicine is selected from barna (Crateva adansonii DC subsp formosensis Jacobs), Da Ye nanmu (Machilus japonica Sieb﹠amp; Zucc var kusanoi (Hayata)), tuber fern (Nephrolepis auriculata (L) Trimen), dewdrop grass (Dichondra micrantha Urban), Roripa montana (Yin Du Han dish) (Rorippa indica (L) Hiern), Ranunculus sceleratus (Ranunculus sceleratusL), Yang Tong (Cleyera japonica Thunb var morii (Yamamoto) Masam), Herba Cayatiae Japonicae (Cayratia japonica (Thunb) Gagnep), Huashan alum (Symplocos chinensis (Lour) Druce), thorn amaranth (Amaranthus spinosus L) Rou Cutters (Lindera akoensis Hayata), chicken mulberry (Morus australis Poir), serration leaf Eurya plant (Eurya nitida Korthals), little red young pearl (Breynia officinalis Hemsley var accrescens (Hayata) MJ Deng ﹠amp; JCWang), callicarpa pedunculata (Taiwan Japanses beauty-berry) (Callicarpa formosana Rolfe), Lanyu Chinese cassia tree (Cinnamomum kotoense Kanehira ﹠amp; Sasaki), smalt (Clerodendrum cyrtophyllumTurcz), Radix zanthoxyli (Zanthoxylum nitidum (Roxb) DC), Chloranthus glaber (Sarcandra glabra (Thunb) Nakai), hairy euphorbia (Chamaesyce hirta (L) Millsp), mochi (milk leaf rattan) (Gonostegia hirta (Blume) Miq), milk banyan (Ficus erecta Thunb var beecheyana (Hook ﹠amp; Arn)), Acanthopanax obovatus Hoo Machilus nanmu (Machilus obovatifolia (Hayata) Kanehira ﹠amp; Sasaki), Rose Mallow Root (Urena lobata L), brick red azalea (Rhododendron oldhamiiMaxim), Taiwan Chinese incense cedar (Calocedrus macrolepis Kurz var formaosana (Florin)), and composition thereof.
The material of plastic slide of the present invention can be homopolymer or multipolymer, it is that to be selected from following monomer prepared by having one or more: ethene, ethylene halide, propylene, halogenation propylene, acrylate, methacrylate, butadiene, vinyl cyanide, norborene and styrene, wherein cinnamic polymkeric substance is preferred.The material of this plastic slide also can be a polycarbonate.The slide glass size that the sizableness of the plastic slide that the present invention is used is used always in microarray device or laser scanner.The advantage of the employed plastic slide of method of the present invention is to use various chemical agent to handle the surface of this plastic slide, make and be not only big molecule (such as protein and DNA), also comprise that micromolecule (such as the metabolin of herbal medicine) can be fixed on the surface of this plastic slide.This advantage seems important especially if contrast glass slide commonly used only can be used for fixing the fact of big molecule (such as protein and DNA).Moreover plastic material can be easy to be molded as required shape, and also has the low superiority of cost.Plastic slide can have one or more grooves, by conditional decisions such as needs, manufacturing cost, detection sensitivities.In preferred system of the present invention, this plastic slide has two grooves, and the ground point sample and can be added to the solution that contains probe on this groove to carry out hybridization reaction on the surface of this groove subsequently but format in the sample hurdle that is obtained from herbal ingredients.The degree of depth of these 2 grooves can be identical or different, and between being low to moderate 0.03 millimeter in up to 0.5 millimeter scope.After molded, relative 2 limits of per 1 groove can have railing respectively to be used for the supporting cover slide in addition, and wherein this cover glass is used to avoid evaporating or loses this and is added to the solution that contains probe to this groove.
The anticipating of this plastic slide can be utilized polyfunctional group aldehyde and be immersed in subsequently provides NH
2In the precursor solution of group, so that the plastic slide that is generated contains active amine in its surface.This provides NH
2The precursor of group can be organism or inorganics, and can be selected from NH
4OH, primary amine, secondary amine or tertiary amine, this primary amine wherein, the aliphatic series of secondary amine and tertiary amine and/or aromatic series partly can be used for useing as extra introns.Provide NH for this
2The precursor of group can directly provide free NH
2The NH of group
4OH is preferred.
In the present invention, the coating on this plastic slide can be made of multifunctional molecule (for example, the multi-group ring oxide), and it plays the effect of introns.This multi-group ring oxide's the effect composition that herbal medicine is contained is connected on this plastic slide through handling in advance.The lip-deep amido reaction of the active epoxy base of this multi-group ring oxide's a end and this plastic slide through handling in advance, and the contained composition of the active epoxy base of this multi-group ring oxide's other end absorption herbal medicine or with the contained composition reaction of herbal medicine.Specifically, the compound that contains free hydroxyl, sulfydryl and/or amido in the herbal medicine composition can form covalent bond with the active epoxy base of this multi-group ring oxide's the other end, thereby this compound can be connected on this plastic slide through applying.This multi-group ring oxide is preferably contained the long chemical chain of 6-24 carbon atom, make the contained composition of herbal medicine can be not directly in conjunction with or be adsorbed onto on this plastic slide through handling in advance.In the present invention, per 1 point sample sample is firm to the combination of the plastic slide through applying, even as the same after rigorous disengaging flushing.Importantly in the present invention, be not only big molecule (such as protein and DNA), also comprise that micromolecule (such as metabolin) can be fixed in the mode of homogeneous phase or non-homogeneous phase on the surface of this plastic slide through applying.
To the classification sample of herbal medicine extract is loaded on step on this plastic slide through applying in the mode of microarray, the classification sample of this herbal medicine extract is fixed on this plastic slide through applying, and wherein each sample spot can contain the herbal medicine composition that is homogeneous phase or non-homogeneous phase.In the method for the invention, can use conformability microminiaturization technology (integrating miniaturization technology), perhaps carry out with manual type to increase the density of hurdle lattice sample on this plastic slide through applying.
In the method for the invention, detect the mode that exists required biological activity to be based on the subject matter guiding with the quality of determining this herbal medicine in the herbal medicine, it comprises that the solution that will contain label probe is loaded on the groove of this plastic slide through applying to carry out hybridization reaction (wherein can utilize glass sheet to cover each groove and contain the evaporation of the solution of label probe to prevent this), and utilize instrument (for example, laser scanner) with video picture and identify the sample spot sampling point that can combine with this label probe or react.The employed probe of method of the present invention is based on the fixed known subject matter that is homogeneous phase or non-homogeneous phase of molecular mechanism, it can be, for example, antagonism is such as cell, acceptor, peptide or the protein-based micromolecular compound selected, competitive part or antibody.The label of this linking probe can be dyestuff or radiomaterial.
Preferred system of the present invention provides a kind of method of definite herb character, and it is to utilize the technology for detection of biochip to go out to be present in the biological activity that can combine with tumor necrosis factor-alpha (TNF-α) selectivity in the herbal medicine.Concrete, carry out hybridization reaction as probe in the method for the invention through the tumor necrosis factor-alpha (TNF-α) of biotin (biotin) mark with through the Streptavidin (strepavidin) of Cy3 mark.The method according to this invention, show the composition and the signal that combines through biotin labeled TNF-α in the sample spot sampling point on the treated plastic slide if observe, it shows in the composition of this sample spot sampling point to have at least a kind of candidate compound, and it has the biologically active that is similar to antagonism TNF-α.These candidate compounds can be used for treating autoimmune disease, such as rheumatoid arthritis.
Need not further work, we believe that all those skilled in the art all can the disclosed content of instructions fully implement all or step partly according to the present invention.Following embodiment only is used to illustrate method of the present invention, never is to limit the scope of the invention.
Embodiment
Carry out according to step shown in Figure 2
The allocation step of herbal medicine extract: with the herbal medicine collected after flushing and drying, 50 restrain add in the herbal medicine methyl alcohol (40/1, v/w).Utilize the following step to extract composition in the herbal medicine: to mix the potpourri of gained, the methanol extraction liquid of gained 8000rpm and 4 ℃ centrifugal 30 minutes down, obtaining supernatant, and is merged.Repeat under these conditions to extract 2 times.Utilize rotary evaporator (Heidolph, Laborota 4000) that the extract that merges is concentrated to about 30 milliliters of final volume.In concentrated extract, add 10 milliliters of ethanol and 10 milliliters of H
2O then goes up violent the mixing 2 minutes in Vortex (Heidolph, Redax top).Then 12000rpm and 4 ℃ down centrifugal 5 minutes to remove insoluble particle/agglomerate.The clarified supernatant of gained is component A.
A component A (100 microlitre) transferred in 0.5 milliliter the micro tube, in each micro tube, add 100 microlitre EtOAc and 100 microlitre H
2O.Violent blend mixture on Votex is then 12000rpm and 4 ℃ centrifugal 5 minutes down.After the layering, isolate organic layer (B component) and water layer (component E) and two 0.5 milliliter the micro tube of then being placed in.Respectively at adding 100 microlitre H in the micro tube that contains organic layer
2O, and adding 100 microlitre EtOAc further distribute in containing the micro tube of water layer, acutely mix on Votex respectively subsequently.Two layers of potpourri of gained 12000rpm and 4 ℃ centrifugal 5 minutes down, are isolated organic layer and water layer respectively to two samples respectively then, altogether four components, be denoted as component C, D, F and G respectively, as shown in Figure 2.
Embodiment 2
Anticipating of plastic slide
Utilization is by the prepared and molded plastic slide of styrene polymer, and it contains two grooves.The size of this molded plastics slide glass is equivalent to microscope or the employed microslide commonly used of laser scanner, and wherein the degree of depth of this groove is 0.05 millimeter.
At first, at room temperature make this molded plastics slide glass be immersed in 0.4% glutaraldehyde solution (pH5.0) and reach 4 hours, wash and under 60 ℃, be soaked in 3M NH with a large amount of water subsequently
4Reach 4 hours in the OH aqueous solution (pH11.0).Under 37 ℃, utilize 100mM 1,4-butanediol diglycidyl ether (pH11.0) is handled the plastic slide that is generated and is spent the night.The plastic slide of gained 0.1M NaHCO
3Aqueous solution (pH8.0) flushing once and with distilled water is washed four times, is stored in the distilled water under 4 ℃ then.Slide glass with gained at room temperature is stored in the safe cupboard before use.
Embodiment 3
Herbal ingredients is added on the treated plastic slide
The component A to G of all embodiment 1 gained is gone up dry at SpeedVac (Savant), and then it is dissolved in the 50mM carbonate buffer solution (pH9.5) that 50 microlitres contain 30%DMSO again.Distribute the artificial point sample of component in the groove of the treated plastic slide of embodiment 2 gained in each herbal medicine, repeat secondary.The volume of each point of sample is the 0.1-0.2 microlitre.Will be following dry 30 minutes at 37 ℃ through the plastic slide of point sample.Then the slide glass with gained is soaked in a large amount of 1M monoethanolamine aqueous solution (pH8.0), then cultivated 2 hours down at 37 ℃, during gentle every now and then the stirring.Wash slide glass three times with TBST damping fluid (containing 50mM Tris-HCl (pH7.3) and 0.15M NaCl and 0.05% Tween 20), wash slide glass three times with a large amount of distilled waters then.Make slide glass place 5 minutes down to dry at 37 ℃.The slide glass of handling through herbal medicine of gained can be used for hybridization reaction or can be at the vacuum lower seal immediately, and 4 ℃ of following long preservation.Before adding the fluorescence labeling probe of using for hybridization reaction, (GenePix 4000, AXON) scanned slide in advance to use laser scanner.
Embodiment 4
The biotinylation reaction of tumor necrosis factor (TNF-α)
The biotinylation reaction of TNF-alpha protein is to use biotin amido group caproic acid N-hydroxy-succinamide ester (biotinamidocaproate N-hydroxysuccinimide ester) (BACHSE, Sigma B-2643) to carry out.Dimethyl sulfoxide (DMSO) solution (5mg/ml) of preparation BACHSE.Be added in the TNF-α solution with the ratio of 1: 40 (w/w) BACHESE solution gained.At room temperature reacted 30 minutes, during stir reactant occasionally.Then reaction mixture under 4 ℃ with distilled water dialysis 1 hour, then with TBST damping fluid dialysis 16-18 hour, during in the TBST secondary that more renews.The biotinylation extent of reaction of TNF-alpha protein is according to the method for knowing in the prior art, utilize 15%SDS-PAGE immunoadsorption experiment (wherein avidin-alkaline phosphatase (avidin-alkaline phosphatase) chemistry covers on the biotin of biotinylated TNF-α) to test, then before using with biotinylated tumor necrosis factor (B-TNF α) in 4 ℃ of following storage vials.
Embodiment 5
With fluorophore (fluorophore) mark streptavidin (strepavidin)
According to the method for manufacturer's suggestion, with FluoriLink
TMCy3
TMDifunctional chemically-reactive dyes (Amersham) mark streptavidin (strepavidin) is with preparation Cy3-streptavidin (SA).Utilizing gel-filtration chromatography to remove unreacted fluorescent dye with the Bio-Gel P2 tubing string (10 centimetres of 0.5 cm x) of TBST damping fluid balance on (Bio-Rad) in advance.Utilize the optical density (OD of UV spectrophotometer measurement 280 nanometers
280), and the ultimate density that estimates through the streptavidin of Cy3 mark is 0.5mg/ml.
Embodiment 6
The hybridization reaction of the slide glass of handling through herbal medicine
At the difference cover glass cover plate (22 millimeters * 22 millimeters) on each groove of the plastic slide that herbal medicine is handled of embodiment 3 gained, the B-TNF α that dilutes by embodiment 4 gained with the TBST damping fluid is that 0.5 mcg/ml obtains working solution to concentration.20-25 microlitre B-TNF α solution is gently injected in each groove of the plastic slide that herbal medicine is handled, slide glass was at room temperature cultivated 2 hours, then with the flushing of TBST damping fluid, then leniently with excessive TBST damping fluid flushing three times with distilled water flushing four times.Slide glass is following dry 5 minutes at 37 ℃.Is that 0.5 mcg/ml prepares fluorescent reagent by diluting by embodiment 5 resulting Cy3-SA mother liquors with the TBST damping fluid to concentration.The fluorescent reagent of 20-25 microlitre is gently injected on each treated plastic slide that contains B-TNF α.The slide glass of gained was at room temperature cultivated 2 hours or was cultivated under 4 ℃ and spend the night.Slide glass is through leniently washing four times with excessive TBST damping fluid flushing three times with distilled water, and is following dry 5 minutes at 37 ℃ then.The slide glass of gained uses GenePix 4000A slide glass scanner (AxonInstruments) scanography under 650 nanometers, utilize GenePix 3.0 software analysis fluorescence spots.The results are shown in Fig. 1.
The result
Prepare each herbal medicine extract according to standard step shown in Figure 2.Preliminary thick mass component is denoted as component A.Then carrying out part respectively by the component A of each herbal medicine gained grade is divided into 6 solution and (is denoted as B component-G) respectively.The herbal medicine of 27 kinds of researchs is shown in the table 1 with its latin name, wherein the 27th kind of herbal medicine and first kind of identical barna (Crateva adansonii DC subsp formosensis Jacobs) that is of herbal medicine, just the concentration of the 27th kind of herbal medicine is two times of first kind of herbal medicine concentration.Fig. 1 is presented at hybridization preceding (road 1) and hybridizes the image that back (road 2) gets via scanned slide.The digital 1-28 that indicates among Fig. 1 is the numbering according to the herbal medicine shown in the table 1.English alphabet A-G is corresponding to other extracted component according to step gained shown in Figure 2.Each extracted component is by two parts of the herbal medicine specimen preparations of correspondence.
The most herbal medicine extracted component of image proof shown in the road 1 has the endogenous fluorophore, and it shows the endogenous red fluorescence under the optical excitation of 650 nano wave lengths.The expression of green fluorescence point on the road 2 of Fig. 1 combines through the B-TNF of Cy3 mark α and herbal medicine extracted component.Endogenous red fluorescence that yellow fluorescence point on the road 2 of Fig. 1 may be interpreted as the herbal medicine extracted component and the mixed effect that has combined through the green fluorescence of the B-TNF of Cy3 mark α.The herbal medicine extracted component of each herbal medicine individuality is summarized in the table 1 with the activity that combines of B-TNF α.The result of table 1 shows that 17 kinds in 27 kinds of herbal medicine have the activity that combines with TNF α in different extracted components.Activity be according at the ratio (R) of the fluorescence intensity of 650 nanometers and 570 nanometers and with ++ ,+or-classify, wherein R 〉=1 is defined as " ", 0.6＜R＜1.0 are defined as "+", reach R≤0.6 and are defined as " ++ ".
Table 1
| The vegetable formal name of the herbal medicine of analyzing | Active with combining of TNF α | |||||||
| A | B | C | D | E | F | G | ||
| 1 | Barna Crateva adansonii DC subsp formosensis Jacobs | - | - | - | - | - | - | - |
| 2 | Da Ye nanmu Machilus japonica Sieb﹠Zucc var kusanoi (Hayata) | ++ | ++ | + | ++ | ++ | - | + |
| 3 | Tuber fern Nephrolepis auriculata (L) Trimen | ++ | ++ | ++ | ++ | ++ | ++ | - |
| 4 | Dewdrop grass Dichondra micrantha Urban | - | - | - | - | - | - | - |
| 5 | Roripa montana (Yin Du Han dish) Rorippa indica (L) Hiern | - | - | - | - | - | - | - |
| 6 | Ranunculus sceleratus Ranunculus sceleratus L | + | + | - | - | - | - | - |
| 7 | Yang Tong Cleyera japonica Thunb var morii (Yamamoto) Masam | + | + | - | - | - | - | - |
| 8 | Herba Cayatiae Japonicae Cayratia japonica (Thunb) Gagnep | + | + | + | + | + | - | - |
| 9 | Huashan alum Symplocos chinensis (Lour) Druce | ++ | ++ | + | ++ | ++ | ++ | ++ |
| 10 | Thorn amaranth Amaranthus spinosus L | + | + | - | + | + | + | - |
| 11 | Rou Cutters Lindera akoensis Hayata | - | - | - | - | - | - | - |
| 12 | Chicken mulberry Morus australis Poir | - | - | - | - | - | - | - |
| 13 | Serration leaf Eurya plant Eurya nitida Korthals | ++ | - | - | + | + | - | - |
| 14 | Little red young pearl Breynia officinalis Hemsley var accrescens (Hayata) MJ Deng ﹠ JC Wang | + | + | + | + | + | + | - |
| 15 | Callicarpa pedunculata (Taiwan Japanses beauty-berry) Callicarpa formosana Rolfe | + | + | + | + | ++ | ++ | + |
| 16 | Lanyu Chinese cassia tree Cinnamomum kotoense Kanehira ﹠ Sasaki | + | + | - | + | - | - | - |
| 17 | Smalt Clerodendrum cyrtophyllum Turcz | - | - | - | - | - | - | - |
| 18 | Radix zanthoxyli Zanthoxylum nitidum (Roxb) DC | - | - | - | - | - | - | - |
| 19 | Chloranthus glaber Sarcandra glabra (Thunb) Nakai | + | + | + | + | + | - | - |
| 20 | Hairy euphorbia Chamaesyce hirta (L) Millsp | + | - | - | + | ++ | + | - |
| 21 | Mochi (milk leaf rattan) Gonostegia hirta (Blume) Miq | - | - | - | - | - | - | - |
| 22 | Milk banyan Ficus erecta Thunb var beecheyana (Hook ﹠ Arn) | - | - | - | - | - | - | - |
| 23 | Acanthopanax obovatus Hoo Machilus nanmu Machilus obovatifolia (Hayata) Kanehira ﹠ Sasaki | - | - | - | - | - | - | - |
| 24 | Taiwan Chinese incense cedar (leaf) Calocedrus macrolepis Kurz var formaosana (Florin) | ++ | + | + | + | - | - | - |
| 25 | Rose Mallow Root Urena lobata L | ++ | ++ | - | ++ | + | + | - |
| 26 | Brick red azalea Rhododendron oldhamii Maxim | + | + | + | + | - | - | - |
| 27 | Barna Crateva adansonii DC subsp formosensis Jacobs | - | - | - | - | - | - | - |
| 28 | Taiwan Chinese incense cedar (stem) Calocedrus macrolepis Kurz var formaosana (Florin) | + | + | + | + | - | + | - |
Claims (37)
1. method of separating and identifying the herbal medicine active ingredient is characterized in that the method comprising the steps of:
(a) obtain the herbal medicine extract with the solvent extraction herbal medicine, then this herbal medicine extract of evaporation and concentration;
(b) the herbal medicine extract that concentrates is mixed mutually with water and water-miscible organic solvent, remove formed insoluble particle then to obtain component A;
(c) component A is mixed with water and water-insoluble organic solvents, and component A is distributed extraction, and layering, organic component B and water component E obtained;
(d) mixed water and said components B and distribute extraction obtain organic component C and water component D;
(e) mix water-insoluble organic solvents and said components E, and distribute extraction, obtain organic component F and water component G;
(f) resulting component A-G is added on the pretreated plastic slide respectively; With
(g) carry out hybridization reaction and input to detect the active ingredient that is present in the herbal medicine extracted component.
2. the method for claim 1 is characterized in that, the ratio of described step (a) Chinese herbal medicine and solvent be 1: 30 to 1: 50w/v.
3. method as claimed in claim 2 is characterized in that, the ratio of described step (a) Chinese herbal medicine and solvent is 1: 40w/v.
4. the method for claim 1 is characterized in that, used solvent is selected from water, organic solvent and combination thereof in the described step (a).
5. method as claimed in claim 4 is characterized in that, used solvent is a polar organic solvent in the described step (a).
6. method as claimed in claim 5 is characterized in that, used solvent is an alkanol in the described step (a).
7. method as claimed in claim 6 is characterized in that, used solvent is the alkanol with 1 to 6 carbon atom in the described step (a).
8. method as claimed in claim 7 is characterized in that, used solvent is a methyl alcohol in the described step (a).
9. the method for claim 1, it is characterized in that, extraction in the described step (b) comprises: mix the resulting herbal medicine extract of described step (a) and water and water-miscible organic solvent, its plant liquid extract: water: the ratio of water-miscible organic solvent is 2-4: 0.5-1.5: 0.5-1.5v: v: v.
10. method as claimed in claim 9, it is characterized in that, extraction in the described step (b) comprises: herbal medicine extract and the water and the water-miscible organic solvent of gained in the blend step (a), its plant liquid extract: water: the ratio of water-miscible organic solvent is 3: 1: 1v: v: v.
11. method as claimed in claim 10 is characterized in that, described insoluble particle utilizes centrifuge method to remove.
12. method as claimed in claim 10 is characterized in that, used water-miscible organic solvent is the alkanol with 1-6 carbon atom in the described step (b).
13. method as claimed in claim 12 is characterized in that, used water-miscible organic solvent is an ethanol in the described step (b).
14. the method for claim 1, it is characterized in that, distribution extraction in the described step (c) comprises: blending ingredients A and water and water-insoluble organic solvents, wherein component A: water: the ratio of water-insoluble organic solvents is 0.5-1.5: 0.5-1.5: 0.5-1.5v: v: v.
15. method as claimed in claim 14 is characterized in that, the distribution extraction in the described step (c) comprises: blending ingredients A and water and water-insoluble organic solvents, wherein component A: water: the ratio of water-insoluble organic solvents is 1: 1: 1v: v: v.
16. method as claimed in claim 14 is characterized in that, described water-insoluble organic solvents is the carboxylate with 3-10 carbon atom.
17. method as claimed in claim 16 is characterized in that, described water-insoluble organic solvents is an ethyl acetate.
18. the method for claim 1 is characterized in that, the blend step in the described step (d) comprises: with B component: the ratio of water is mode blending ingredients B and the water of 0.5-1.5: 0.5-1.5v/v.
19. method as claimed in claim 18 is characterized in that, the blend step in the described step (d) comprises: with B component: the ratio of water is 1: mode blending ingredients B and the water of 1v/v.
20. the method for claim 1 is characterized in that, the blend step in the described step (e) comprises: with the ratio of water-insoluble organic solvents: component E is that the mode of 0.5-1.5: 0.5-1.5v/v is mixed water-insoluble organic solvents and component E.
21. method as claimed in claim 20 is characterized in that, the blend step in the described step (e) comprises with water-insoluble organic solvents: the ratio of component E is 1: the mode of 1v/v is mixed water-insoluble organic solvents and component E.
22. method as claimed in claim 20 is characterized in that, used water-insoluble organic solvents is an ethyl acetate in the described step (e).
23. the method for claim 1, it is characterized in that, the material of this plastic slide is a polycarbonate, or by one or more monomers prepared homopolymer or multipolymer, described monomer is selected from: ethene, ethylene halide, propylene, halogenation propylene, acrylate, methacrylate, butadiene, vinyl cyanide, norborene and styrene.
24. method as claimed in claim 23 is characterized in that, this plastic slide is made with styrene polymer.
25. the method for claim 1 is characterized in that, described anticipate be included in apply this plastic slide before, this plastic slide is handled and is soaked in subsequently through polyfunctional group aldehyde earlier provides NH
2In the precursor solution of group, and then apply multi-functional molecule formation coat at this plastic cement slide glass.
26. method as claimed in claim 25 is characterized in that, described polyfunctional group aldehyde is glutaraldehyde.
27. method as claimed in claim 25 is characterized in that, the described NH that provides
2The precursor of group is NH
4OH.
28. method as claimed in claim 25 is characterized in that, this multifunctional molecule is the multi-group ring oxide of containing at least 1 epoxy radicals at each end.
29. method as claimed in claim 28 is characterized in that, the epoxy radicals of this multi-group ring oxide's a end and this lip-deep amido reaction through pretreated plastic slide.
30. method as claimed in claim 28 is characterized in that, the epoxy radicals of this multi-group ring oxide's the other end and the free hydroxyl of the composition in the herbal medicine, sulfhydryl or amido reaction.
31. method as claimed in claim 28 is characterized in that, this multi-group ring oxide is contained the chemical chain of the length with 6-24 carbon atom.
32. the method for claim 1 is characterized in that, this herbal medicine contains at least a composition that can combine with tumor necrosis factor-alpha (TNF-α) selectivity.
33. the method for claim 1 is characterized in that, is added to each component on the plastic slide and is the component from a kind of herbal medicine in step (f), or from the potpourri of the corresponding component of at least a herbal medicine.
34. method as claimed in claim 33, it is characterized in that, described herbal medicine is selected from: barna, Da Ye nanmu, tuber fern, dewdrop grass, Roripa montana (seal degree Han dish), Ranunculus sceleratus, Yang Tong, Herba Cayatiae Japonicae, Huashan alum, thorn amaranth, meat Cutters, chicken mulberry, serration leaf Eurya plant, little red young pearl, callicarpa pedunculata (Taiwan Japanses beauty-berry), Lanyu Chinese cassia tree, smalt, Radix zanthoxyli, Chloranthus glaber, hairy euphorbia, mochi (milk leaf rattan), milk banyan, Acanthopanax obovatus Hoo Machilus nanmu, Rose Mallow Root, brick red azalea, Taiwan Chinese incense cedar, and composition thereof.
35. the method for claim 1 is characterized in that, the solution that this method utilization contains label probe carries out hybridization reaction.
36. method as claimed in claim 35 is characterized in that, the described solution that contains label probe is homogeneous phase or homogeneous phase not.
37. method as claimed in claim 36 is characterized in that, described mark is dyestuff or radiomaterial.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02124697 CN1219214C (en) | 2002-06-21 | 2002-06-21 | Novel process for determining herb character |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02124697 CN1219214C (en) | 2002-06-21 | 2002-06-21 | Novel process for determining herb character |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1464305A CN1464305A (en) | 2003-12-31 |
| CN1219214C true CN1219214C (en) | 2005-09-14 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 02124697 Expired - Fee Related CN1219214C (en) | 2002-06-21 | 2002-06-21 | Novel process for determining herb character |
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| Country | Link |
|---|---|
| CN (1) | CN1219214C (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8225458B1 (en) | 2001-07-13 | 2012-07-24 | Hoffberg Steven M | Intelligent door restraint |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112924669B (en) * | 2021-02-08 | 2021-11-19 | 南京农业大学 | Multicolor flow measurement immunochromatography test strip based on three primary colors of optics and preparation and detection methods thereof |
-
2002
- 2002-06-21 CN CN 02124697 patent/CN1219214C/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8225458B1 (en) | 2001-07-13 | 2012-07-24 | Hoffberg Steven M | Intelligent door restraint |
| US9045927B1 (en) | 2001-07-13 | 2015-06-02 | Steven M. Hoffberg | Intelligent door restraint |
| US9121217B1 (en) | 2001-07-13 | 2015-09-01 | Steven M. Hoffberg | Intelligent door restraint |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1464305A (en) | 2003-12-31 |
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