CN102288712A - Method for simultaneous determination of piperine content and piperlonguminine content in long piper extract through HPLC (high performance liquid chromatography) - Google Patents
Method for simultaneous determination of piperine content and piperlonguminine content in long piper extract through HPLC (high performance liquid chromatography) Download PDFInfo
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Abstract
The invention discloses a method for simultaneous determination of the piperine content and the piperlonguminine content in long piper extract through HPLC (high performance liquid chromatography). The mobile phase of HPLC adopts mixture of methanol and formic acid solution of 0.2-0.3 percent (the volume percentage content), wherein the volume ratio of the formic acid solution and methanol is (32-37):(62-67). The piperine content and the piperlonguminine content in the long piper extract are simultaneously determined for the first time through HPLC in the experiment, the method for quality control of the long piper extract is further improved, and the contents of long piper extracts in different batches can be determined. Compared with the methodological survey model of the traditional external standard method for the piperine, the method complies with the multi-component and multi-target thinking mode in traditional Chinese medicine, and thus is more objective and scientific.
Description
Technical field
The present invention relates to simultaneously content assaying method to pipering in the Bi roots of grass and two kinds of compositions of the bright peaceful alkali of the Bi roots of grass.
Background technology
The Bi roots of grass is the dry immature fruit ear of the Piperaceae Piperaceae Piper Piper plant Bi roots of grass (Piper longum L.), in being, cover, hide, dimension doctor's custom medication, has warming spleen and stomach for dispelling cold, the function of promoting qi circulation and relieving pain is recorded in " Chinese pharmacopoeia (version was an one in 2010) and " Pharmacopoeia of People's Republic of China the Sanitation Ministry medicine standard Uygur medicine fascicle ".Chemical constitution study for the Bi roots of grass shows that major ingredients is alkaloid and amide-type composition in the Bi roots of grass, contains terpene, other constituents of volatilization wet goods in addition.Modern pharmacology studies show that, the Bi roots of grass has widely pharmacologically active such as anti-gastrointestinal tract disease, anti-inflammation, anti-oxidant etc.It is that index components is carried out quality control to Bi roots of grass medicinal material that 2010 editions pharmacopeia and relevant document are selected the active component pipering for use.But ingredient complexity in the Chinese medicine, clinical efficacy is the combined action of multiple composition, so establish unique effective constituent of medical drugs in a certain composition conduct certain limitation is arranged.And select for use a plurality of effective constituents could reflect important inherent quality more really simultaneously as the important quantitative index.Just controlling the pattern of drug quality according to a plurality of content of effective, is present stage to one of focus of the method for quality control research of Chinese medicine and compound thereof.The bright peaceful alkali of the Bi roots of grass also is the effective constituent in the Bi roots of grass, has effects such as platelet aggregation-against.In recent years, the bright peaceful alkali of the Bi roots of grass is more and more paid close attention to owing to its clear and definite pharmacological action is subjected to people.Therefore, two kinds of compositions of pipering in the Bi roots of grass and the bright peaceful alkali of the Bi roots of grass are seemed particularly important as the index of the quality control of the Bi roots of grass simultaneously.
Summary of the invention
The object of the present invention is to provide a kind of method of measuring pipering and the bright peaceful alkali content of the Bi roots of grass in the plant simultaneously.
Method provided by the invention is measured detected sample by high performance liquid chromatography, the moving phase of described high performance liquid chromatography is methyl alcohol and (0.2-0.3) mixed liquor formed of the aqueous formic acid of % (volumn concentration), and the volume ratio of described aqueous formic acid and described methyl alcohol is (32-37): (62-67).
The volume ratio of above-mentioned aqueous formic acid and methyl alcohol can be 35: 65; The volumn concentration of formic acid specifically can be 0.25% in the described aqueous formic acid.
The chromatographic column that above-mentioned high performance liquid chromatography adopts is C18; The flow velocity of moving phase is (0.8-1.2) mLmin
-1, be preferably 1.0mLmin
-1The detection wavelength is 343nm; Column temperature is 38-42 ℃, is preferably 40 ℃; Sample size is 8-12ul, is preferably 10ul.
Further, above-mentioned plant is the Bi roots of grass, and described detected sample is a Bi roots of grass extract.
Above-mentioned Bi roots of grass extract obtains as follows: with Bi roots of grass extract ultrasonic Extraction, described extract is preferably methyl alcohol.
The ratio of the said extracted liquid and the Bi roots of grass can be (25-250) ml: (0.05-0.15) g; Be preferably (25-250) ml: (0.09-0.11); 100ml: 0.1g more preferably.
The extraction time of above-mentioned ultrasonic Extraction can be 8-30 minute, and preferably 10-30 minute, more preferably 10 minutes; The power of described ultrasonic Extraction is 40-50kHz, preferably 45kHz.
The above-mentioned Bi roots of grass is through pulverization process, and described pulverization process is that the Bi roots of grass is pulverized was the Bi roots of grass powder of 80 mesh sieves.
The preparation method of the used bright peaceful alkali reference substance of the Bi roots of grass of basis of calculation curve comprises the steps: in the above-mentioned high performance liquid chromatography
1) with Bi roots of grass medicinal material alcohol extract, collect filtrate, remove ethanol, obtain total extract;
2) with described total extract dichloromethane extraction, collect extraction phase, remove methylene chloride, obtain dichloromethane extract;
3) with described dichloromethane extract volume ratio be 1: 1 methylene chloride-methanol dissolving, with 100-200 order silica gel mixed sample, through silica gel column chromatography, the eluent that obtains 0-2000ml with the methylene chloride wash-out as the eluent of first component and 2000-2500ml as second component, collect second component, remove methylene chloride, obtain dry thing after the drying;
4) the dry thing with step 3) is dissolved in the methyl alcohol, on the C18 post, be that 5: 5 methanol-water wash-outs obtain component 1-component 10 totally ten components with volume ratio, be that 5.5: 4.5 methanol-water wash-outs obtain component 11-component 18 totally eight components with volume ratio again, be that 6: 4 methanol-water wash-outs obtain component 19-component 23 with volume ratio then, each component 200ml in the step 4), collect component 23, remove the first alcohol and water, obtain the bright peaceful alkali reference substance of the Bi roots of grass.
In the step 1), the number of times of described alcohol extract is 2 times; Ethanol is that percent by volume is 85% ethanol water; The proportioning of described Bi roots of grass medicinal material and described ethanol water is (2-4) kg: (12-15) L, preferably 3kg: (12-15) L.
Step 2) in, the number of times of described dichloromethane extraction is 3 times, and the volume ratio of described methylene chloride and described total extract is 5: 1.
In the step 3), the high 60cm of the silica gel in the silicagel column, silicagel column internal diameter 4.0cm.
In the step 4), described C18 post is to be 5: 5 methanol-water pre-equilibration through volume ratio; The high 80cm of silica gel in the C18 post, column internal diameter 1.5cm.
Above-mentioned method also belongs to protection scope of the present invention in the application of identifying or control in the Bi roots of grass quality of medicinal material.
Above-mentioned evaluation or control Bi roots of grass quality of medicinal material are preferably realized by measuring the bright peaceful alkali content of the pipering and the Bi roots of grass simultaneously.
The application of above-mentioned method in the bright peaceful alkali of the preparation Bi roots of grass also belongs to protection scope of the present invention.
The application that above-mentioned method is measured in pipering and the bright peaceful alkali content of the Bi roots of grass at the same time also belongs to protection scope of the present invention.
Experimental results show that: ultrasonic method is the highest to the extraction ratio of pipering in the medicinal material and the bright peaceful alkali of the Bi roots of grass, and the running time is shorter relatively, and is comparatively simple and convenient, is the optimum extracting method (table 1) of need testing solution preparation so select ultrasonic method.Ultrasonic Extraction 10min can more fully extract pipering in the Bi roots of grass medicinal material and the bright peaceful alkali of the Bi roots of grass, saves for considering cost, so finally to select with methyl alcohol ultrasonic Extraction 10min be that need testing solution prepares optimum extracting method (table 2).100mL methyl alcohol can more fully extract pipering in the Bi roots of grass medicinal material and the bright peaceful alkali of the Bi roots of grass, saves for considering cost, so finally to select with 100mL methyl alcohol ultrasonic Extraction be that need testing solution prepares optimum extracting method (table 3).After adopting HPLC condition of the present invention and above-mentioned extraction conditions, the RSD of pipering and the bright peaceful alkali day to day precision of the Bi roots of grass is respectively 1.0% and 0.5%, all less than 3.0%, shows precision good (table 6,7); The RSD of pipering and the bright peaceful alkali of the Bi roots of grass is respectively 1.4% and 1.2%, all less than 3%.The result shows need testing solution in 72h stable (table 8) at least; The bright peaceful alkali massfraction RSD value of the pipering and the Bi roots of grass is 2.9%, repeatability fine (table 10); The average recovery rate of pipering and RSD are respectively 100.4% and 2.5%, and the average recovery rate and the RSD of the bright peaceful alkali of the Bi roots of grass are respectively 102.1% and 2.0%, and average recovery meet the requirements (table 11,12) is described.
Therefore the content that the HPLC method is measured pipering and the bright peaceful alkali of the Bi roots of grass in the Bi roots of grass is simultaneously set up in this experiment first, the further perfect method of quality control of the Bi roots of grass, and the Bi roots of grass of different lot numbers carried out assay.Compare with the methodological study model of in the past pipering external standard method, more meet the thoughtcast thereby more objective, the science of " multicomponent " in motherland's traditional medicine, " many target spots ".
Description of drawings
The bright peaceful alkali of Fig. 1 Bi roots of grass
1H-NMR collection of illustrative plates 1.
The bright peaceful alkali of Fig. 2 Bi roots of grass
1H-NMR collection of illustrative plates 2.
The bright peaceful alkali of Fig. 3 Bi roots of grass
13The C-NMR collection of illustrative plates.
Fig. 4 pipering typical curve.
The bright peaceful alkali typical curve of Fig. 5 Bi roots of grass.
The bright peaceful alkali standard items liquid chromatogram of Fig. 6 pipering and the Bi roots of grass.
Fig. 7 Bi roots of grass medicinal material need testing solution (lot number: 200805) high-efficient liquid phase chromatogram.
Fig. 8 Bi roots of grass medicinal material need testing solution (lot number: 200905) high-efficient liquid phase chromatogram.
Fig. 9 Bi roots of grass medicinal material need testing solution (lot number: 201005) high-efficient liquid phase chromatogram.
Figure 10 Bi roots of grass medicinal material need testing solution (lot number: 200810) high-efficient liquid phase chromatogram.
Figure 11 Bi roots of grass medicinal material need testing solution (lot number: 201003) high-efficient liquid phase chromatogram.
Figure 12 Bi roots of grass medicinal material need testing solution (lot number: 201008) high-efficient liquid phase chromatogram.
Figure 13 Bi roots of grass medicinal material need testing solution (lot number: 201102) high-efficient liquid phase chromatogram.
Figure 14 Bi roots of grass medicinal material need testing solution (lot number: 201103) high-efficient liquid phase chromatogram.
Embodiment
The invention will be further described below in conjunction with specific embodiment, but the present invention is not limited to following examples.
Among the following embodiment, if no special instructions, be conventional method.
Agilent1100 type high performance liquid chromatograph series, comprise: G1365D DAD (detecting device), G1311A QuatPump (quaternary gradient pump), G1332A (online vacuum degassing machine), G1329A ALS (automatic sampler), G1316AColcom (column oven), Agilent Chemstation system (workstation) (Agilent Technologies, the U.S.); ME235S 100,000/electronic analytical balance (Sai Duolisi scientific instrument company limited), KQ5200DV type numerical control supersonic washer (40kHz, Kunshan Ultrasonic Instruments Co., Ltd.); Agilent Eclipse XDB-C18 chromatographic column (250mm * 4.6mm, 5 μ m) (Agilent Technologies company, the U.S.) pipering standard items (middle inspection institute, lot number 0775-200203, purity＞99%), (embodiment 1 step 1 and two are seen in the self-control of traditional Chinese medicine institute of the Capital University of Medical Sciences to the bright peaceful alkali standard items of the Bi roots of grass, purity＞99%), the Bi roots of grass medicinal material of different lot numbers is 8 batches.Bi roots of grass medicinal material powder (end) among the present invention is the Bi roots of grass powder that Bi roots of grass pulverizing medicinal materials was become 80 mesh sieves.
One, the preparation of extract in the Bi roots of grass
Bi roots of grass medicinal material 3kg pulverizes the back and adds 15L 85% (percent by volume) ethanol water, and reflux 1.5h filters reservation filtrate, filter residue adds the ethanol water backflow 1.5h of 12L 85% (percent by volume), filter, merge filtrate twice, concentrating and obtaining not having pure concentrate is 1950mL.
With the dichloromethane extraction of 5 times of volumes of concentrate three times, obtain the about 43g of dichloromethane extract powdered extract after reclaiming solvent.
Two, the preparation (gradient elution) of the bright peaceful alkali reference substance of the Bi roots of grass
The medicinal extract methylene chloride: methyl alcohol (1: 1) is dissolving fully, with 100-200 order silica gel mixed sample, through silicagel column (the high 60cm of silica gel, column internal diameter 4.0cm) chromatogram, with methylene chloride-methanol (volume ratio is 10: 0～9: 1～8: 2) gradient elution.Be divided into Fr.1～8 eight part, wherein obtain Fr1 (elution volume 2000ml), Fr2 (elution volume 500ml), Fr3 (elution volume 1000ml), Fr4 (elution volume 1000ml) with 10: 0 wash-outs of methylene chloride-methanol; Obtain Fr5 (elution volume 500ml), Fr6 (elution volume 1000ml), Fr7 (elution volume 750ml) with 9: 1 wash-outs of methylene chloride-methanol; Obtain Fr8 (elution volume 1250ml) with 8: 2 wash-outs of methylene chloride-methanol, reclaim solvent, wherein Fr.2 drying is weighed as 2.6g.
Fr.2 is dissolved in the small amount of methanol, C18 silica gel chromatographic column (the high 80cm of silica gel, column internal diameter 1.5cm) is through methanol-water (5: 5) 400mL balance, wet method application of sample, with methanol-water (volume ratio is 5: 5～5.5: 4.5～6: 4～6.5: 3.5) gradient elution, collect each concentration gradient eluent.Receive wash-out stream part by every part of 200mL, must flow a part Fr.1～38 respectively, wherein obtain Fr1-Fr10, obtain Fr11-Fr18, obtain Fr19-Fr24 with 6: 4 wash-outs of methanol-water with 5.5: 4.5 wash-outs of methanol-water with 5: 5 wash-outs of methanol-water, obtain Fr28-Fr38 with 6.5: 3.5 wash-outs of methanol-water, dissolve with small amount of methanol behind each stream part evaporated under reduced pressure solvent.Wherein Fr.23 is the white solid powder, is pure simplification compound after testing, it is carried out magnetic resonance spectroscopy measure.
Spectral data is as follows:
1H-NMR (MeOD, 600MHz): δ 6.11 (1H, d, J=14.74Hz), 7.27 (1H, m), 6.79 (1H, m), 6.82 (1H, m), 7.07 (1H, d, J=1.35Hz), 6.80 (1H, d, J=8.03Hz), 0.93 (3H, d, J=6.70Hz), 5.96 (2H, s), 3.09 (2H, t, J=6.48Hz), 1.82 (1H, m), 0.93 (3H, d, J=6.70Hz), 6.95 (1H, dd, J=1.34,8.03Hz).
13C?NMR(MeOD，150MHz)6：169.3，123.9，142.3，126.2，140.3，132.6，106.9，150.0，109.6，1244，102.9，20.7，30.0，20.7，48.3。See Fig. 1～3.Determine that with the document contrast this compound is the bright peaceful alkali of the Bi roots of grass.Detecting its purity through HPLC is 99%, is the bright peaceful alkali reference substance of the Bi roots of grass.
Three, the investigation of Bi roots of grass medicinal material extract method
1. the selection of extracting method
Following three groups of experiments are set determine best extracting method:
1) ultrasonic extraction: get the about 0.1g of Bi roots of grass medicinal powder, the accurate title, decide, and puts in the 100mL volumetric flask, adds methyl alcohol 100mL, close plug, ultrasonic (45kHz) extracts 30min, puts coldly, complements to scale with methyl alcohol, shake up, filter, get subsequent filtrate, promptly get need testing solution with 0.45 μ m miillpore filter.
2) reflux extraction: get the about 0.1g of Bi roots of grass medicinal powder, the accurate title, decide, and places the 100mL round-bottomed flask, adds methyl alcohol 100mL, claim to decide weight, reflux 1h is put coldly, weighs again, supply the weight that subtracts mistake with methyl alcohol, shake up with the filtration of 0.45 μ m miillpore filter, get subsequent filtrate, promptly get need testing solution.
3) soxhlet extraction: get the about 0.1g of Bi roots of grass medicinal powder, the accurate title, decide, and places the 100mL conical flask, adds methyl alcohol 100mL, behind the cold soaking 24h, shakes up, and filters with 0.45 μ m miillpore filter, gets subsequent filtrate, promptly gets need testing solution.
The test sample of above-mentioned three kinds of Different Extraction Method gained is carried out the HPLC detection.Each sample introduction 10 μ L of above need testing solution measure each peak area value, represent wherein pipering and the bright peaceful alkali content of the Bi roots of grass with peak area.Testing conditions is: chromatographic column: Agilent Technologies company, Agilent Eclipse XDB-C18 chromatographic column (250mm * 4.6mm, 5 μ m); Moving phase: methyl alcohol-volume ratio 0.25% aqueous formic acid (65: 35); Flow velocity: 1.0mLmin
-1Detect wavelength: 343nm; Column temperature: 40 ℃.
Table 1 Different Extraction Method is investigated the result
The result is as shown in table 1, and ultrasonic method is the highest to the extraction ratio of pipering in the medicinal material and the bright peaceful alkali of the Bi roots of grass, and the running time is shorter relatively, and is comparatively simple and convenient, is the optimum extracting method of need testing solution preparation so select ultrasonic method.
2, the selection of extraction time
On the basis of step 1, the experiment of different extraction times is set:
Precision takes by weighing 3 parts of Bi roots of grass medicinal powders, and each 0.1g splits in the 100mL volumetric flask, add methyl alcohol 100mL, close plug, ultrasonic (45kHz) handles 10min, 20min, 30min respectively, put coldly, complement to scale, shake up with methyl alcohol, filter with 0.45 μ m miillpore filter, get subsequent filtrate, promptly get the need testing solution of different extraction times, all each sample introduction 10 μ L, adopt HPLC (testing conditions is identical with step 1) to measure each peak area value, represent wherein pipering and the bright peaceful alkali content of the Bi roots of grass with peak area.
The different extraction times investigation of table 2 result
The result is as shown in table 2, and ultrasonic Extraction 10min can more fully extract pipering in the Bi roots of grass medicinal material and the bright peaceful alkali of the Bi roots of grass, saves for considering cost, so finally to select with methyl alcohol ultrasonic Extraction 10min be that need testing solution prepares optimum extracting method.
3. extraction solvent volume
On step 1,2 basis, different experiments of extracting solvent volume are set:
Precision takes by weighing 4 parts of Bi roots of grass medicinal powders, each 0.1g, place 25mL respectively, 50mL, 100mL is in the 250mL volumetric flask, add methanol constant volume to scale, close plug, ultrasonic (45kHz) handles 10min respectively, puts cold, complement to scale with methyl alcohol, shake up, filter, get subsequent filtrate with 0.45 μ m miillpore filter, promptly get the need testing solution of different extraction times, all each sample introduction 10 μ L adopts HPLC (testing conditions is identical with step 1) to measure each peak area value, represents wherein pipering and the bright peaceful alkali content of the Bi roots of grass with peak area.
The different solvents that extract of table 3 are investigated the result
The result is as shown in table 3, and 100mL methyl alcohol can more fully extract pipering in the Bi roots of grass medicinal material and the bright peaceful alkali of the Bi roots of grass, saves for considering cost, so finally to select with 100mL methyl alcohol ultrasonic Extraction be that need testing solution prepares optimum extracting method.
Four, measure the foundation and the detection thereof of pipering and the bright peaceful alkali content assay method of the Bi roots of grass in the Bi roots of grass simultaneously
1, the preparation of pipering and the bright peaceful alkali typical curve of the Bi roots of grass
Precision takes by weighing pipering 4.04mg, places the 50mL volumetric flask, adds dissolve with methanol and is diluted to scale, shake up, as the pipering storing solution, the accurate successively 1mL that draws, 2mL, 3mL, 4mL, 5mL, 6mL, 7mL, 8mL place the 10mL volumetric flask respectively, after adding methanol constant volume, promptly get serial pipering reference substance solution; Precision takes by weighing the bright peaceful alkali standard items 1.02mg of the above-mentioned Bi roots of grass, places the 25mL volumetric flask, adds methanol constant volume and is configured to 0.0408mgmL
-1The bright peaceful alkaline reserve liquid of the Bi roots of grass, dilution is made into 0.001671mgmL successively
-1, 0.002089mgmL
-1, 0.002611mgmL
-1, 0.003264,0.00408mgmL
-1, 0.00612mgmL
-1, 0.00816mgmL
-1Standard solution.Above-mentioned reference substance solution is sample introduction 10 μ L respectively, adopt HPLC to measure each peak area value (the results are shown in Table 4, table 5), are horizontal ordinate with reference substance solution concentration, and peak area is an ordinate, and the drawing standard curve (is seen Fig. 4, Fig. 5).The regression equation that gets pipering is Y=63629X-11.219, and r=0.9999 shows that pipering is at 0.00808-0.06464mgmL
-1Scope in be good linear relationship; The bright peaceful alkali regression equation of the Bi roots of grass is Y=61187X-19.452, and r=0.9999 shows that the bright peaceful alkali of the Bi roots of grass is at 0.00167-0.00816mgmL
-1Scope in be good linear relationship.Pipering and the bright peaceful alkali lye of the Bi roots of grass chromatogram are mutually seen Fig. 6.
The standard curve determination data of table 4 pipering
The bright peaceful alkali standard curve determination data of the table 5 Bi roots of grass
2. the configuration of need testing solution
Take by weighing Bi roots of grass medicinal powder 0.1g, place the 100mL volumetric flask, add methanol constant volume to scale, close plug, respectively ultrasonic (45kHz) handles 10min, puts coldly, complements to scale with methyl alcohol, shakes up, filter with 0.45 μ m miillpore filter, get subsequent filtrate, promptly get the need testing solution of Bi roots of grass medicinal material.
3. precision experiment
Drawing above-mentioned concentration is 0.03232mgmL
-1Pipering standard solution and above-mentioned 0.02089mgmL
-1The bright peaceful alkali standard solution of the Bi roots of grass under the chromatographic condition of step 31, repeats sample introduction 5 times on the same day, the peak area of record pipering and the bright peaceful alkali of the Bi roots of grass is withinday precision (seeing Table 6).The withinday precision RSD of pipering and the bright peaceful alkali of the Bi roots of grass is respectively 0.6% and 0.3%; With above-mentioned need testing solution continuous sample introduction 3 days, every duplicate samples sample introduction 3 pins, record peak area were day to day precision (seeing Table 7).The RSD of pipering and the bright peaceful alkali day to day precision of the Bi roots of grass is respectively 1.0% and 0.5%, all less than 3.0%, shows that precision is good.
Table 6 withinday precision measurement result (n=5)
Table 7 day to day precision measurement result (n=5)
4. stability experiment
Get Bi roots of grass sample 0.1g, prepare need testing solution Bi roots of grass need testing solution according to method in the step 42, room temperature is placed 72h, respectively 0,2,4,6,8,10h, 12h, 24h, 48h, 72h utilize and carry out the HPLC inspection under the chromatographic condition of step 31 and know, and the record peak area, see Table 8, table 9.The RSD of pipering and the bright peaceful alkali of the Bi roots of grass is respectively 1.4% and 1.2%, all less than 3%.The result shows that need testing solution is stable in 72h at least.
Table 8 pipering stability experiment data
The bright peaceful alkaline stability experimental data of the table 9 Bi roots of grass
5. repeated experiment
Precision takes by weighing Bi roots of grass medicinal powder 0.1g, according to 5 parts of Bi roots of grass need testing solutions of method preparation in the step 42, carries out the HPLC inspection and know under the chromatographic condition of step 31, and the peak area of the pipering of gained and the bright peaceful alkali of the Bi roots of grass sees Table 10.The bright peaceful alkali massfraction RSD value of the pipering and the Bi roots of grass is respectively 2.9% and 2.9%.
Table 10 repeated experiment measurement result (n=5)
6. average recovery experiment
6 parts of the Bi roots of grass medicinal powders that takes by weighing, every part of 0.1g, it is an amount of to add the pipering standard items respectively, presses need testing solution preparation method operation, and 31 chromatographic conditions are measured set by step, and measurement result sees Table 11; 6 parts of the Bi roots of grass medicinal powders that takes by weighing, every part of 0.2g, it is an amount of to add the bright peaceful alkali standard items of the Bi roots of grass respectively, presses need testing solution preparation method operation, and measures by above-mentioned chromatographic condition, and measurement result sees Table 12.The average recovery rate of pipering and RSD are respectively 100.4% and 2.5%, and the average recovery rate and the RSD of the bright peaceful alkali of the Bi roots of grass are respectively 102.1% and 2.0%.
Table 11 pipering average recovery measurement result
The bright peaceful alkali average recovery measurement result of the table 12 Bi roots of grass
7. the assay of Bi roots of grass medicinal material
Take by weighing 8 crowdes of Bi roots of grass medicinal powder 0.1g that the source is different respectively, according to 8 parts of need testing solutions of method preparation in the step 42,31 chromatographic condition is measured set by step.The results are shown in Table 13, Fig. 7～Figure 14.
The massfraction (n=3) of pipering and the bright peaceful alkali of the Bi roots of grass in the Bi roots of grass medicinal material in the different lot numbers of table 13
Because the complicacy and the diversity of traditional Chinese medicine ingredients aspect the quality control of Chinese medicine, are tended to multicomponent, many control indexes at present.And only adopt pipering for the quality control of the Bi roots of grass in the past is single index components.This experiment has broken through that only to adopt pipering be the single index method of quality control of index components in the past, measure the content of pipering and the bright peaceful alkali of the Bi roots of grass in the Bi roots of grass simultaneously, for the foundation of many index ' s qualities of Bi roots of grass medicinal material control criterion provides foundation, also provide reference for other medicinal material quantitative test that contains this type of amide alkaloid constituents.
In order to improve the extraction ratio of analytic target, reduce the interference of impurity, this research is the extraction solvent with methyl alcohol, is index with the extraction ratio of pipering and the bright peaceful alkali of the Bi roots of grass, has compared cold soaking extraction method, heating and refluxing extraction method and ultrasonic extraction respectively.The result shows, reflux extraction and ultrasonic extraction ratio are suitable, all are better than cold-maceration.Consider that ultrasonic extraction is simple to operation, extraction time is short, controlled, the leaching process favorable reproducibility of each operating parameter, help to realize the high reappearance of collection of illustrative plates, therefore select ultrasonic extraction.And investigated extraction time respectively, extracted the influence of solvent volume to extraction ratio, the result shows the liquid material than 100mL: 0.1g, and ultrasonic Extraction 10min can extract analytic target fully.
Claims (10)
1. method of measuring pipering and the bright peaceful alkali content of the Bi roots of grass in the plant simultaneously, detected sample is measured by high performance liquid chromatography, the moving phase of described high performance liquid chromatography is methyl alcohol and (0.2-0.3) mixed liquor formed of the aqueous formic acid of % (volumn concentration), and the volume ratio of described aqueous formic acid and described methyl alcohol is (32-37): (62-67).
2. the method for claim 1, it is characterized in that: the volume ratio of described aqueous formic acid and methyl alcohol is 35: 65; The volumn concentration of formic acid is 0.25% in the described aqueous formic acid.
3. method as claimed in claim 1 or 2 is characterized in that: the chromatographic column that described high performance liquid chromatography adopts is C18; The flow velocity of moving phase is (0.8-1.2) mLmin
-1, be preferably 1.0mLmin
-1The detection wavelength is 343nm; Column temperature is 38-42 ℃, is preferably 40 ℃; Sample size is 8-12ul, is preferably 10ul.
4. as arbitrary described method among the claim 1-3, it is characterized in that: described plant is the Bi roots of grass, and described detected sample is a Bi roots of grass extract; Described Bi roots of grass extract obtains as follows: with Bi roots of grass extract ultrasonic Extraction, described extract is preferably methyl alcohol.
5. as arbitrary described method among the claim 1-4, it is characterized in that: the ratio of the described extract and the Bi roots of grass is (25-250) ml: (0.05-0.15) g; Be preferably (25-250) ml: (0.09-0.11); 100ml: 0.1g more preferably.
6. as arbitrary described method among the claim 1-5, it is characterized in that: the extraction time of described ultrasonic Extraction is 8-30 minute, preferably 10-30 minute, and more preferably 10 minutes; The power of described ultrasonic Extraction is 40-50kHz, preferably 45kHz.
7. as arbitrary described method among the claim 4-6, it is characterized in that: the described Bi roots of grass is through pulverization process, and described pulverization process is that the Bi roots of grass is pulverized was the Bi roots of grass powder of 80 mesh sieves.
8. as arbitrary described method among the claim 1-7, it is characterized in that: the preparation method of the used bright peaceful alkali reference substance of the Bi roots of grass of basis of calculation curve comprises the steps: in the described high performance liquid chromatography
1) with Bi roots of grass medicinal material alcohol extract, collect filtrate, remove ethanol, obtain total extract;
2) with described total extract dichloromethane extraction, collect extraction phase, remove methylene chloride, obtain dichloromethane extract;
3) with described dichloromethane extract volume ratio be 1: 1 methylene chloride-methanol dissolving, with 100-200 order silica gel mixed sample, through silica gel column chromatography, the eluent that obtains 0-2000ml with the methylene chloride wash-out as the eluent of first component and 2000-2500ml as second component, collect second component, remove methylene chloride, obtain dry thing after the drying;
4) the dry thing with step 3) is dissolved in the methyl alcohol, on the C18 post, be that 5: 5 methanol-water wash-outs obtain component 1-component 10 totally ten components with volume ratio, be that 5.5: 4.5 methanol-water wash-outs obtain component 11-component 18 totally eight components with volume ratio again, be that 6: 4 methanol-water wash-outs obtain component 19-component 23 with volume ratio then, each component 200ml in the step 4), collect component 23, remove the first alcohol and water, obtain the bright peaceful alkali reference substance of the Bi roots of grass.
9. method as claimed in claim 8 is characterized in that: in the step 1), the number of times of described alcohol extract is 2 times; Ethanol is that percent by volume is 85% ethanol water; The proportioning of described Bi roots of grass medicinal material and described ethanol water is (2-4) kg: (12-15) L, preferably 3kg: (12-15) L;
Step 2) in, the number of times of described dichloromethane extraction is 3 times, and the volume ratio of described methylene chloride and described total extract is 5: 1;
In the step 3), the high 60cm of the silica gel in the silicagel column, silicagel column internal diameter 4.0cm;
In the step 4), described C18 post is to be 5: 5 methanol-water pre-equilibration through volume ratio; The high 80cm of silica gel in the C18 post, column internal diameter 1.5cm.
10. the method described in the claim 1-9 is in control Bi roots of grass quality of medicinal material or in the bright peaceful alkali of the preparation Bi roots of grass or measure application in pipering and the bright peaceful alkali content of the Bi roots of grass at the same time.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104807938A (en) * | 2015-05-04 | 2015-07-29 | 李焕婷 | Mongolian medicine long pepper medicine efficacy material basis analysis method |
| CN104815011A (en) * | 2015-05-04 | 2015-08-05 | 薄彧坤 | Mongolian medicine piper longum extract used for reducing blood fat |
| CN113702520A (en) * | 2021-08-17 | 2021-11-26 | 中南民族大学 | Method for controlling quality of long pepper medicinal material |
| US11485725B2 (en) | 2017-12-15 | 2022-11-01 | Auransa Inc. | Derivatives of piperlongumine and uses thereof |
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| JPS5842686A (en) * | 1981-09-08 | 1983-03-12 | T Hasegawa Co Ltd | Extraction of anti-oxidizing component in natural pepper |
| CN101863872A (en) * | 2010-06-05 | 2010-10-20 | 内蒙古医学院 | Method for extracting PiPerlonguminine and drug combination comprising same |
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| JPS5842686A (en) * | 1981-09-08 | 1983-03-12 | T Hasegawa Co Ltd | Extraction of anti-oxidizing component in natural pepper |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104807938A (en) * | 2015-05-04 | 2015-07-29 | 李焕婷 | Mongolian medicine long pepper medicine efficacy material basis analysis method |
| CN104815011A (en) * | 2015-05-04 | 2015-08-05 | 薄彧坤 | Mongolian medicine piper longum extract used for reducing blood fat |
| US11485725B2 (en) | 2017-12-15 | 2022-11-01 | Auransa Inc. | Derivatives of piperlongumine and uses thereof |
| CN113702520A (en) * | 2021-08-17 | 2021-11-26 | 中南民族大学 | Method for controlling quality of long pepper medicinal material |
| CN113702520B (en) * | 2021-08-17 | 2022-09-16 | 中南民族大学 | Method for controlling quality of long pepper medicinal material |
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