CN102809617B - Herba houttuyniae aboveground part extract and detection method thereof - Google Patents
Herba houttuyniae aboveground part extract and detection method thereof Download PDFInfo
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Abstract
The invention provides an aboveground part extract and a detection method thereof. The extract is detected via an HPLC (High Performance Liquid Chromatography) fingerprint method. The herba houttuyniae aboveground part extract covers multi-class active components, is unambiguous is material basis, can serve as a new standard control for medicinal material quality detection, and more facilitates quality control to the herba houttuyniae aboveground part. According to the invention, the optimal detection method of the herba houttuyniae aboveground part is achieved via a comprehensively surveying sample solution preparation method and color spectrum conditions. When the detection method is applied, a base line is stable, chromatographic peak shape and resolution are favorable, and meanwhile, flavonoid and organic acid chemical compounds of the herba houttuyniae can be synchronously detected, provides a reliable method for quality detection of the herba houttuyniae aboveground part, and ensures medicinal material quality of the herba houttuyniae aboveground.
Description
Technical field
The present invention relates to a kind of detection method of cordate houttuynia aerial part.
Background technology
Cordate houttuynia, is fresh herb or the dry aerial parts of saururaceae plant houttuynia cordata Houttuynia cordata Thunb., and its taste is pungent, and cold nature is returned lung channel, can be clearing heat and detoxicating, the carbuncle that disappears apocenosis, inducing diuresis for treating strangurtia, is used for the treatment of lung carbuncle pyemesis, phlegm heat panting is coughed, hot dysentery, and heat is drenched, carbuncle sore tumefacting virus.
Pharmacological research discovery, cordate houttuynia has stronger antibacterial action to the various bacteria such as staphylococcus aureus, shigella dysenteriae; Cordate houttuynia decoction has inhibiting effect to virus, its fixed oil part, obviously flu-prevention virus; Cordate houttuynia decoction can also strengthen leukocytic phagocytic activity, has immunological enhancement; Contained quercitin energy nephrectasia blood vessel in cordate houttuynia, improves renal blood flow, may be relevant to the diuresis of cordate houttuynia.The current research to cordate houttuynia finds, it is relevant to its medical active that its aerial part contains more chemical composition, as volatile oil: decanoyl acetaldehyde, lauryl aldehyde, australene, linalool, cloves alkene, Bronyl acetate etc.; Flavonoids: Hyperoside, rutin, quercitin, isoquercitrin etc.; Organic acid: chlorogenic acid, linoleic acid etc. (" Chinese medicine voluminous dictionary " 2006 editions).
Research for cordate houttuynia aerial part and under ground portion is also found, in aerial part, Hyperoside, quercitin equal size are apparently higher than under ground portion (Zheng Yimin, Deng, the content of Hyperoside and quercitin in Houttuynia cordata by HPLC, R&D of modern TCM and practice, the 3rd phase of 19 volumes in 2005).The above-mentioned result of study to chemical composition content, has illustrated that the medical active of cordate houttuynia aerial part is more good; Meanwhile, also point out that its dry aerial parts can be separately as Herba Houttuyniae source in pharmacopeia, this medical value that cordate houttuynia aerial part has also been described is higher, corresponding with the result of study of above-mentioned chemical composition content.
In order effectively to control the quality of Herba Houttuyniae, at present existing researchist adopts efficient liquid-phase chromatograph finger print atlas to analyze the chemical composition of cordate houttuynia aerial part, as Lu Hongmei etc., take methyl alcohol-phosphate buffer system as mobile phase, 50% ethanol extract to cordate houttuynia aerial part has carried out HPLC detection, and measure the content (Lu Hongmei of rutin, quercitin and 3 kinds of flavones ingredients of Quercetin, Deng, the efficient liquid-phase chromatograph finger print atlas analysis of flavones ingredient in cordate houttuynia, chromatogram, 28 10 phases of volume in 2010).But, in this detection method, being to measure after certain density alcohol extract medicinal material, and in present agent, being mostly to adopt decocting form to extract cordate houttuynia, gained composition and 50% ethanol extract have different; Simultaneously, in the method, only there is flavone compound for the index of evaluating quality of medicinal material, and other chemical compositions in cordate houttuynia also have good medical active (as organic acid compounds), therefore, only take flavone compound as evaluation index, treatment that can not complete reaction Herba Houttuyniae.
Therefore, being badly in need of a kind of can be the method that detects all sidedly cordate houttuynia aerial part quality.
Meanwhile, in the time using liquid phase to detect Chinese crude drug quality, be mostly to adopt reference substance or finger-print to detect at present, wherein, while adopting reference substance to detect, can not fully reflect the quality of medicinal material; And adopt when finger-print, although can react quality of medicinal material comprehensively,, also may be because the difference of the many factors such as detecting instrument, mobile phase, environmental baseline, cause the error of measurement.If a kind of standard extract of medicinal material can be provided, in this extract, comprise more effective constituent, and material base is clear and definite, so, this extract is combined after use with finger-print detection means, must avoid or reduce the error that extraneous factor is brought, more can effectively reflect quality of medicinal material.
Summary of the invention
The object of the present invention is to provide a kind of cordate houttuynia overground part extract that is more conducive to cordate houttuynia aerial part to carry out quality control; Another object of the present invention is to provide a kind of detection method of cordate houttuynia aerial part.
The invention provides a kind of cordate houttuynia overground part extract, feature in the HPLC finger-print of this extract is as follows: have 6 characteristic peaks, take the retention time of quercitin chromatographic peak and peak area as reference, calculate relative retention time and the relative peak area at other peaks, wherein
No. 1 peak: relative retention time is 0.224-0.273, relative peak area is 0.26-1.02;
No. 2 peaks: relative retention time is 0.300-0.366, relative peak area is 0.26-1.05;
No. 3 peaks: relative retention time is 0.351-0.429, relative peak area is 0.12-0.48;
No. 4 peaks: relative retention time is 0.824-0.870, relative peak area is 0.34-1.37;
No. 5 peaks: relative retention time is 0.843-0.890, relative peak area is 0.06-0.26;
The relative retention time of quercitin chromatographic peak is 1, and relative peak area is 1;
The chromatographic condition of above-mentioned HPLC finger-print is as follows:
Chromatographic column: make filling agent with octadecylsilane chemically bonded silica;
Detect wavelength: 254 ± 5nm;
Mobile phase: take 0.1-0.5%V/V glacial acetic acid solution as mobile phase A, acetonitrile is Mobile phase B, carries out gradient elution by following program:
Since 0 minute to 15 minutes, mobile phase A became 89% from 95%, since 15 minutes to 45 minutes, mobile phase A becomes 87% from 89%, and since 45 minutes to 60 minutes, mobile phase A became 65% from 87%, since 60 minutes to 63 minutes, mobile phase A became 95% from 65%.
Further, the feature of described finger-print is as follows: there are 6 characteristic peaks, take the retention time of quercitin chromatographic peak and peak area as reference, calculate relative retention time and the relative peak area at other peaks, wherein,
No. 1 peak: relative retention time 0.24-0.25, accounts for the Area Ratio 0.4-0.6 with reference to peak;
No. 2 peak: relative retention time 0.33-0.34, account for the Area Ratio 0.4-0.6 with reference to peak;
No. 3 peak: relative retention time 0.39-0.40, account for the Area Ratio 0.2-0.4 with reference to peak;
No. 4 peak: relative retention time 0.84-0.85, account for the Area Ratio 0.6-0.8 with reference to peak;
No. 5 peak: relative retention time 0.86-0.87, account for the Area Ratio 0.1-0.2 with reference to peak;
The relative retention time of quercitin chromatographic peak is 1, and relative peak area is 1.
Further, the feature of described finger-print is as follows: there are 6 characteristic peaks, take the retention time of quercitin chromatographic peak and peak area as reference, calculate relative retention time and the relative peak area at other peaks, wherein,
No. 1 peak: relative retention time 0.248, accounts for the Area Ratio 0.51 with reference to peak;
No. 2 peaks: relative retention time 0.333, accounts for the Area Ratio 0.52 with reference to peak;
No. 3 peaks: relative retention time 0.392, accounts for the Area Ratio 0.24 with reference to peak;
No. 4 peaks: relative retention time 0.848, accounts for the Area Ratio 0.68 with reference to peak;
No. 5 peaks: relative retention time 0.867, accounts for the Area Ratio 0.13 with reference to peak;
The relative retention time of quercitin chromatographic peak is 1, and relative peak area is 1.
Further preferably, described standard finger-print as shown in Figure 4.
The present invention also provides a kind of detection method of cordate houttuynia aerial part, and it adopts HPLC finger print method to detect, and it comprises following operation steps:
(1) preparation of need testing solution:
Get cordate houttuynia aerial part, supernatant after water extract-alcohol precipitation, remove after ethanol, upper nonpolar or low pole large pore resin absorption column, first washes with water to water lotion colourless, use again 50%～90%V/V ethanol elution, collect ethanol eluate, except after desolventizing, residue dissolves with 50%～90%V/V methyl alcohol, filter, obtain need testing solution;
(2) chromatographic condition:
Chromatographic column: make filling agent with octadecylsilane chemically bonded silica;
Detect wavelength: 254 ± 5nm;
Mobile phase: take 0.1-0.5%V/V glacial acetic acid solution as mobile phase A, acetonitrile is Mobile phase B, carries out gradient elution by following program:
Since 0 minute to 15 minutes, mobile phase A became 89% from 95%, since 15 minutes to 45 minutes, mobile phase A becomes 87% from 89%, and since 45 minutes to 60 minutes, mobile phase A became 65% from 87%, since 60 minutes to 63 minutes, mobile phase A became 95% from 65%, and maintains 10 minutes;
(3) need testing solution gained chromatogram and standard finger-print are compared, calculate similarity.
Further, in step (1), upper D101 large pore resin absorption column.
Further, in step (1), wash-out is 70%V/V by the concentration of ethanol, and methanol concentration is 70%V/V.
Further, described water extract-alcohol precipitation refers to: by after the boiling of cordate houttuynia aerial part, the clear cream that when decocting liquid is concentrated into 60～80 ℃, relative density is 1.10～1.30, adds ethanol to being 50%～90%V/V containing alcohol amount, leaves standstill.
Further, in step (2), described mobile phase A is 0.2%V/V glacial acetic acid solution.
Further, in step (2), detection wavelength is 254nm.
Further, described chromatographic column is Inertsil ODS-3 150 × 4.6mm, 5 μ m.
Further, in step (2), column temperature is 35 ℃, and flow velocity is 1.0ml/min.
Further, in described HPLC finger print method, take a kind of compound in quercitin, Hyperoside, chlorogenic acid or two or more potpourri as reference substance.
Quercitin and Hyperoside are flavones ingredient, and chlorogenic acid is organic acid compound.Pharmacological research is found at present, and Hyperoside, has the effect of good treatment acute and chronic tracheitis, asthma, and the effects such as damage, Myocardial Lipid Peroxidation in addition resist myocardial ischemia; Quercitin, has stronger antivirus action; Chlorogenic acid, has the effects such as antibacterial, antiviral, anti-oxidant, anti-mutagenesis, antiplatelet are solidified, hepatic cholagogic.The conventional drug activity of the pharmacological action of above-mentioned three kinds of compositions and cordate houttuynia is closely related.Therefore,, if take above-mentioned three components as reference substance, be more conducive to the quality control to cordate houttuynia aerial part.
Further, described cordate houttuynia aerial part is the fresh aerial part of saururaceae plant houttuynia cordata Houttuynia cordata Thunb..
Further, in the method, also comprise the steps:
Wherein, the feature of described standard finger-print is as follows: there are 6 characteristic peaks, take the retention time of quercitin chromatographic peak and peak area as reference, calculate relative retention time and the relative peak area at other peaks, wherein,
No. 1 peak: relative retention time is 0.224-0.273, relative peak area is 0.26-1.02;
No. 2 peaks: relative retention time is 0.300-0.366, relative peak area is 0.26-1.05;
No. 3 peaks: relative retention time is 0.351-0.429, relative peak area is 0.12-0.48;
No. 4 peaks: relative retention time is 0.824-0.870, relative peak area is 0.34-1.37;
No. 5 peaks: relative retention time is 0.843-0.890, relative peak area is 0.06-0.26;
The relative retention time of quercitin chromatographic peak is 1, and relative peak area is 1.
Further, the feature of described standard finger-print is as follows: there are 6 characteristic peaks, take the retention time of quercitin chromatographic peak and peak area as reference, calculate relative retention time and the relative peak area at other peaks, wherein,
No. 1 peak: relative retention time 0.24-0.25, accounts for the Area Ratio 0.4-0.6 with reference to peak;
No. 2 peak: relative retention time 0.33-0.34, account for the Area Ratio 0.4-0.6 with reference to peak;
No. 3 peak: relative retention time 0.39-0.40, account for the Area Ratio 0.2-0.4 with reference to peak;
No. 4 peak: relative retention time 0.84-0.85, account for the Area Ratio 0.6-0.8 with reference to peak;
No. 5 peak: relative retention time 0.86-0.87, account for the Area Ratio 0.1-0.2 with reference to peak;
The relative retention time of quercitin chromatographic peak is 1, and relative peak area is 1.
Further, the feature of described standard finger-print is as follows: there are 6 characteristic peaks, take the retention time of quercitin chromatographic peak and peak area as reference, calculate relative retention time and the relative peak area at other peaks, wherein,
No. 1 peak: relative retention time 0.248, accounts for the Area Ratio 0.51 with reference to peak;
No. 2 peaks: relative retention time 0.333, accounts for the Area Ratio 0.52 with reference to peak;
No. 3 peaks: relative retention time 0.392, accounts for the Area Ratio 0.24 with reference to peak;
No. 4 peaks: relative retention time 0.848, accounts for the Area Ratio 0.68 with reference to peak;
No. 5 peaks: relative retention time 0.867, accounts for the Area Ratio 0.13 with reference to peak;
The relative retention time of quercitin chromatographic peak is 1, and relative peak area is 1.
Further preferably, described standard finger-print as shown in Figure 4.
The cordate houttuynia overground part extract that the present invention prepares, has contained multiclass active component, and its material base is comparatively clear and definite, can be used as a kind of new standard control thing in quality of medicinal material detection, is more conducive to the quality control to cordate houttuynia aerial part.
The present invention, by the integrated survey to need testing solution preparation method, chromatographic condition, has finally obtained best cordate houttuynia aerial part detection method.While utilizing the inventive method to detect, baseline is steady, each chromatogram peak-to-peak shape and degree of separation are all good, and, flavonoids to cordate houttuynia aerial part, organic acid compound detect simultaneously, from the cordate houttuynia aerial part in 13 batches of different places of production, draw the product general character of this medicinal material---be standard finger-print, for the quality testing of cordate houttuynia aerial part provides reliable method, for the quality of medicinal material of cordate houttuynia aerial part provides guarantee.
Accompanying drawing explanation
Fig. 1 is with reference to product collection of illustrative plates (quercitin)
Fig. 2 Hyperoside reference substance collection of illustrative plates
Fig. 3 chlorogenic acid reference substance collection of illustrative plates
Fig. 4 fresh fish raw meat grass aerial part reference fingerprint
The investigation collection of illustrative plates of Fig. 5 mobile phase condition 2
The investigation collection of illustrative plates of Fig. 6 mobile phase condition 3
The investigation collection of illustrative plates of Fig. 7 mobile phase condition 4
The investigation collection of illustrative plates of Fig. 8 chromatographic column condition 2
The investigation collection of illustrative plates of Fig. 9 chromatographic column condition 3
Figure 10 does not cross the investigation collection of illustrative plates of post sample
3 batches of fresh cordate houttuynia sample aerial part finger-prints of Figure 111
Embodiment
The preparation of embodiment 1 cordate houttuynia overground part extract of the present invention
Get the aerial part of fresh cordate houttuynia, twice of boiling, each 0.5 hour, collecting decoction, filtered, it is 1.18～1.20(60～80 ℃ that filtrate is concentrated into relative density) clear cream, add ethanol to being 70% containing alcohol amount, stir evenly, leave standstill 24 hours, filter, decompression filtrate recycling ethanol is also concentrated into appropriate (containing raw medicinal herbs 5g/ml).Get D101 macroporous absorbent resin (internal diameter 1cm on 5ml solution, long 10cm), absorption, washes with water to colourless, then uses 70% ethanol elution to colourless, collect 70% ethanol eluate, evaporate to dryness in water-bath, residue dissolves with 70% methyl alcohol, filters, reclaim after solvent, obtain cordate houttuynia overground part extract of the present invention.
The detection method of embodiment 2 cordate houttuynia aerial part of the present invention
(1) reference substance solution preparation
Get quercitin reference substance appropriate, accurately weighed, add methyl alcohol and make every 1ml containing the solution of 0.1633mg, as with reference to product solution.
Get respectively Hyperoside, chlorogenic acid reference substance is appropriate, accurately weighed, add methyl alcohol and make every 1ml containing Hyperoside 0.0881mg, containing the solution of chlorogenic acid 0.1157mg, product solution in contrast.
(2) need testing solution preparation
Get the aerial part of fresh cordate houttuynia, twice of boiling, each 0.5 hour, collecting decoction, filtered, it is 1.18～1.20(60～80 ℃ that filtrate is concentrated into relative density) clear cream, add ethanol to being 70% containing alcohol amount, stir evenly, leave standstill 24 hours, filter, decompression filtrate recycling ethanol is also concentrated into appropriate (containing raw medicinal herbs 5g/ml).Get D101 macroporous absorbent resin (internal diameter 1cm on 5ml solution, long 10cm), absorption, wash with water to colourless, then use 70% ethanol elution to colourless, collect 70% ethanol eluate, evaporate to dryness in water-bath, residue dissolves with 70% methyl alcohol and is settled in 25ml volumetric flask, shakes up, and crosses the miillpore filter of 0.45 μ m as the need testing solution of fresh fish raw meat grass aerial part.
(3) detection method
Accurate each reference substance solution and the each 10 μ l of need testing solution of drawing respectively, injection liquid chromatography, its chromatographic condition is as follows:
Chromatographic column: Inertsil ODS-3150 × 4.6mm, 5 μ m
Column temperature: 35 ℃
Flow velocity: 1.0ml/min
Detect wavelength: 254nm
Mobile phase: take 0.2% glacial acetic acid solution as mobile phase A, acetonitrile is Mobile phase B, carries out gradient elution by following program: since 0 minute to 15 minutes, mobile phase A became 89% from 95%, since 15 minutes to 45 minutes, mobile phase A becomes 87% from 89%, and since 45 minutes to 60 minutes, mobile phase A became 65% from 87%, since 60 minutes to 63 minutes, mobile phase A becomes 95% from 65%, and maintains 10 minutes, and gradient is linear gradient.
According to said method, take 13 batches of cordate houttuynia sample aerial parts as basis, press chromatographic fingerprints of Chinese materia medica evaluation system, draw the standard finger-print of cordate houttuynia aerial part, as shown in Figure 4, in this finger-print, there are 6 characteristic peaks, take the retention time of quercitin chromatographic peak and peak area as reference, calculate relative retention time and the relative peak area at other peaks, wherein
No. 1 peak: relative retention time 0.248, accounts for the Area Ratio 0.51 with reference to peak;
No. 2 peaks: relative retention time 0.333, accounts for the Area Ratio 0.52 with reference to peak;
No. 3 peaks: relative retention time 0.392, accounts for the Area Ratio 0.24 with reference to peak;
No. 4 peaks: relative retention time 0.848, accounts for the Area Ratio 0.68 with reference to peak;
No. 5 peaks: relative retention time 0.867, accounts for the Area Ratio 0.13 with reference to peak;
The relative retention time of quercitin chromatographic peak is 1, and relative peak area is 1.
Need testing solution gained chromatogram and standard finger-print are compared, calculate similarity.If similarity more than 0.90, shows cordate houttuynia aerial part to be measured and meets examination criteria.
The investigation of embodiment 3 detection methods of the present invention
(1) investigation of chromatographic condition
(1) shaker test of mobile phase
1. reference substance solution preparation
Get quercitin reference substance appropriate, accurately weighed, add methyl alcohol and make every 1ml containing the solution of 0.1633mg, as with reference to product solution.
Get respectively Hyperoside, chlorogenic acid reference substance is appropriate, accurately weighed, add methyl alcohol and make every 1ml containing Hyperoside 0.0881mg, containing the solution of chlorogenic acid 0.1157mg, product solution in contrast.
2. need testing solution preparation
Get the aerial part of fresh cordate houttuynia, twice of boiling, each 0.5 hour, collecting decoction, filtered, it is 1.18～1.20(60～80 ℃ that filtrate is concentrated into relative density) clear cream, add ethanol to being 70% containing alcohol amount, stir evenly, leave standstill 24 hours, filter, decompression filtrate recycling ethanol is also concentrated into appropriate (containing raw medicinal herbs 5g/ml).Get D101 macroporous absorbent resin (internal diameter 1cm on 5ml solution, long 10cm), absorption, wash with water to colourless, then use 70% ethanol elution to colourless, collect 70% ethanol eluate, evaporate to dryness in water-bath, residue dissolves with 70% methyl alcohol and is settled in 25ml volumetric flask, shakes up, and crosses the miillpore filter of 0.45 μ m as the need testing solution of fresh fish raw meat grass aerial part.
3. mobile phase conditional filtering
Condition 1: take 0.2% glacial acetic acid solution as mobile phase A, acetonitrile is Mobile phase B, carries out gradient elution by following program: since 0 minute to 15 minutes, mobile phase A became 89% from 95%, since 15 minutes to 45 minutes, mobile phase A becomes 87% from 89%, and since 45 minutes to 60 minutes, mobile phase A became 65% from 87%, since 60 minutes to 63 minutes, mobile phase A becomes 95% from 65%, and maintains 10 minutes, and gradient is linear gradient.
Condition 2: take 0.2% glacial acetic acid solution as mobile phase A, acetonitrile is Mobile phase B, carries out gradient elution by following program: since 0 minute to 10 minutes, mobile phase A became 90% from 95%, since 10 minutes to 40 minutes, mobile phase A becomes 85% from 90%, and since 40 minutes to 55 minutes, mobile phase A became 65% from 85%, since 55 minutes to 60 minutes, mobile phase A becomes 95% from 65%, and maintains 10 minutes, and gradient is linear gradient.
Condition 3: take 0.2% glacial acetic acid solution as mobile phase A, acetonitrile is Mobile phase B, carries out gradient elution by following program: since 0 minute to 20 minutes, mobile phase A became 85% from 95%, since 20 minutes to 50 minutes, mobile phase A becomes 83% from 85%, and since 50 minutes to 60 minutes, mobile phase A became 65% from 83%, since 60 minutes to 65 minutes, mobile phase A becomes 95% from 65%, and maintains 10 minutes, and gradient is linear gradient.
Condition 4:0.1% phosphoric acid solution is mobile phase A, methyl alcohol is Mobile phase B, carries out gradient elution by following program: since 0 minute to 10 minutes, mobile phase A became 70% from 80%, and maintain 10 minutes, since 20 minutes to 50 minutes, mobile phase A became 35% from 70%, and maintains 5 minutes, since 55 minutes to 56 minutes, mobile phase A becomes 80% from 35%, and maintains 10 minutes, and gradient is linear gradient.
Instrument: Waters2695-2996 high performance liquid chromatograph, Empower chromatographic work station.
Chromatographic column: Inertsil ODS-3(150mm × 4.6mm, 5um), 35 ℃ of column temperatures.
4. assay method and result:
Accurate each reference substance solution and the each 10 μ l of need testing solution of drawing respectively, injection liquid chromatography, UV-detector detects, and mensuration wavelength is 254nm, records chromatogram, observes the separation case of chromatographic peak, and result is referring to Fig. 4-7.
Result shows, mobile phase condition 1 separation the best.
(2) shaker test of chromatographic column
1. reference substance solution preparation
Get quercitin reference substance appropriate, accurately weighed, add methyl alcohol and make every 1ml containing the solution of 0.1633mg, as with reference to product solution.
Get respectively Hyperoside, chlorogenic acid reference substance is appropriate, accurately weighed, add methyl alcohol and make every 1ml containing Hyperoside 0.0881mg, containing the solution of chlorogenic acid 0.1157mg, product solution in contrast.
2. need testing solution preparation
Get the aerial part of fresh cordate houttuynia, twice of boiling, each 0.5 hour, collecting decoction, filtered, it is 1.18～1.20(60～80 ℃ that filtrate is concentrated into relative density) clear cream, add ethanol to being 70% containing alcohol amount, stir evenly, leave standstill 24 hours, filter, decompression filtrate recycling ethanol is also concentrated into appropriate (containing raw medicinal herbs 5g/ml).Get D101 macroporous absorbent resin (internal diameter 1cm on 5ml solution, long 10cm), absorption, wash with water to colourless, then use 70% ethanol elution to colourless, collect 70% ethanol eluate, evaporate to dryness in water-bath, residue dissolves with 70% methyl alcohol and is settled in 25ml volumetric flask, shakes up, and crosses the miillpore filter of 0.45 μ m as the need testing solution of fresh fish raw meat grass aerial part.
3. chromatographic column kind
Condition 1: chromatographic column: Inertsil ODS-3 150mm × 4.6mm, 5um
Condition 2: chromatographic column: Alltech C18 150 × 4.6mm, 5 μ m
Condition 3: chromatographic column: Waters C18 150 × 4.6mm, 5 μ m
Instrument Waters2695-2996 high performance liquid chromatograph, Empower chromatographic work station.
Mobile phase: take 0.2% glacial acetic acid solution as mobile phase A, acetonitrile is Mobile phase B, carries out gradient elution by following program: since 0 minute to 15 minutes, mobile phase A became 89% from 95%, since 15 minutes to 45 minutes, mobile phase A becomes 87% from 89%, and since 45 minutes to 60 minutes, mobile phase A became 65% from 87%, since 60 minutes to 63 minutes, mobile phase A becomes 95% from 65%, and maintains 10 minutes, and gradient is linear gradient.
4. assay method and result:
Accurate each reference substance solution and the each 10 μ l of need testing solution of drawing respectively, injection liquid chromatography, UV-detector detects, and mensuration wavelength is 254nm, records chromatogram, observes the separation case of chromatographic peak, and result is referring to Fig. 4,8,9.
Result shows, chromatographic column condition 1 separation the best.
(3) the investigation result of large pore resin absorption column processing sample
Carry out upper prop wash-out by test sample preparation method in embodiment 1, with the sample sample introduction respectively of wash-out not, chromatographic condition is as embodiment 1, and its result is referring to Fig. 4,10:
Figure 10 is the chromatogram of upper prop not, with Fig. 4 comparison, has the material large compared with multipolarity not easily separated before 10 minutes, so the chromatogram (Fig. 4) after upper prop is more suitable for the quality control of finger-print in retention time.
Result shows, good separation after upper prop wash-out.
(2) instrument precision test
1. chromatographic condition
Chromatographic column: Inertsil ODS-3 150 × 4.6mm, 5 μ m
Column temperature: 35 ℃
Flow velocity: 1.0ml/min
Detect wavelength: 254nm
Mobile phase: take 0.2% glacial acetic acid solution as mobile phase A, acetonitrile is Mobile phase B, carries out gradient elution by following program: since 0 minute to 15 minutes, mobile phase A became 89% from 95%, since 15 minutes to 45 minutes, mobile phase A becomes 87% from 89%, and since 45 minutes to 60 minutes, mobile phase A became 65% from 87%, since 60 minutes to 63 minutes, mobile phase A becomes 95% from 65%, and maintains 10 minutes, and gradient is linear gradient.
2. reference substance solution preparation
Get quercitin reference substance appropriate, accurately weighed, add methyl alcohol and make every 1ml containing the solution of 0.1633mg, as with reference to product solution.
Get respectively Hyperoside, chlorogenic acid reference substance is appropriate, accurately weighed, add methyl alcohol and make every 1ml containing Hyperoside 0.0881mg, containing the solution of chlorogenic acid 0.1157mg, product solution in contrast.
3. need testing solution preparation
Get the aerial part of fresh cordate houttuynia, twice of boiling, each 0.5 hour, collecting decoction, filtered, it is 1.18～1.20(60～80 ℃ that filtrate is concentrated into relative density) clear cream, add ethanol to being 70% containing alcohol amount, stir evenly, leave standstill 24 hours, filter, decompression filtrate recycling ethanol is also concentrated into appropriate (containing raw medicinal herbs 5g/ml).Get D101 macroporous absorbent resin (internal diameter 1cm on 5ml solution, long 10cm), absorption, wash with water to colourless, then use 70% ethanol elution to colourless, collect 70% ethanol eluate, evaporate to dryness in water-bath, residue dissolves with 70% methyl alcohol and is settled in 25ml volumetric flask, shakes up, and crosses the miillpore filter of 0.45 μ m as the need testing solution of fresh fish raw meat grass aerial part.
4. determination method
Accurate reference substance solution and the each 10 μ l of need testing solution of drawing respectively, injection liquid chromatography, UV-detector detects, and mensuration wavelength is 254nm, records chromatogram.Top, fresh fish raw meat meadow divides sample introduction to measure 9 times, calculates the collection of illustrative plates of 9 mensuration and the similarity of reference fingerprint, and record is as following table.
The mensuration precision test of table 1 fresh fish raw meat grass aerial part
(3) stability test
1. chromatographic condition
Chromatographic column: Inertsil ODS-3 150 × 4.6mm, 5 μ m
Column temperature: 35 ℃
Flow velocity: 1.0ml/min
Detect wavelength: 254nm
Mobile phase: take 0.2% glacial acetic acid solution as mobile phase A, acetonitrile is Mobile phase B, carries out gradient elution by following program: since 0 minute to 15 minutes, mobile phase A became 89% from 95%, since 15 minutes to 45 minutes, mobile phase A becomes 87% from 89%, and since 45 minutes to 60 minutes, mobile phase A became 65% from 87%, since 60 minutes to 63 minutes, mobile phase A becomes 95% from 65%, and maintains 10 minutes, and gradient is linear gradient.
2. reference substance solution preparation
Get quercitin reference substance appropriate, accurately weighed, add methyl alcohol and make every 1ml containing the solution of 0.1633mg, as with reference to product solution.
Get respectively Hyperoside, chlorogenic acid reference substance is appropriate, accurately weighed, add methyl alcohol and make every 1ml containing Hyperoside 0.0881mg, containing the solution of chlorogenic acid 0.1157mg, product solution in contrast.
3. need testing solution preparation
Get the aerial part of fresh cordate houttuynia, twice of boiling, each 0.5 hour, collecting decoction, filtered, it is 1.18～1.20(60～80 ℃ that filtrate is concentrated into relative density) clear cream, add ethanol to being 70% containing alcohol amount, stir evenly, leave standstill 24 hours, filter, decompression filtrate recycling ethanol is also concentrated into appropriate (containing raw medicinal herbs 5g/ml).Get D101 macroporous absorbent resin (internal diameter 1cm on 5ml solution, long 10cm), absorption, wash with water to colourless, then use 70% ethanol elution to colourless, collect 70% ethanol eluate, evaporate to dryness in water-bath, residue dissolves with 70% methyl alcohol and is settled in 25ml volumetric flask, shakes up, and crosses the miillpore filter of 0.45 μ m as the need testing solution of fresh fish raw meat grass aerial part.
4. determination method
Accurate reference substance solution and the each 10 μ l of need testing solution of drawing respectively, respectively at 0 hour, 4 hours, 8 hours, 12 hours, 16 hours, 24 hours injection liquid chromatographies, UV-detector detects, and mensuration wavelength is 254nm, records chromatogram.Fresh fish raw meat grass aerial part, at above-mentioned time point sample introduction, calculates the collection of illustrative plates of 6 mensuration and the similarity of reference fingerprint, and record is as following table.
The stability test that table 2 fresh fish raw meat grass aerial part is measured
(4) reappearance test
1. chromatographic condition
Chromatographic column: Inertsil ODS-3 150 × 4.6mm, 5 μ m
Column temperature: 35 ℃
Flow velocity: 1.0ml/min
Detect wavelength: 254nm
Mobile phase: take 0.2% glacial acetic acid solution as mobile phase A, acetonitrile is Mobile phase B, carries out gradient elution by following program: since 0 minute to 15 minutes, mobile phase A became 89% from 95%, since 15 minutes to 45 minutes, mobile phase A becomes 87% from 89%, and since 45 minutes to 60 minutes, mobile phase A became 65% from 87%, since 60 minutes to 63 minutes, mobile phase A becomes 95% from 65%, and maintains 10 minutes, and gradient is linear gradient.
2. reference substance solution preparation
Get quercitin reference substance appropriate, accurately weighed, add methyl alcohol and make every 1ml containing the solution of 0.1633mg, as with reference to product solution.
Get respectively Hyperoside, chlorogenic acid reference substance is appropriate, accurately weighed, add methyl alcohol and make every 1ml containing Hyperoside 0.0881mg, containing the solution of chlorogenic acid 0.1157mg, product solution in contrast.
3. need testing solution preparation
Get the aerial part of fresh cordate houttuynia, twice of boiling, each 0.5 hour, collecting decoction, filtered, it is 1.18～1.20(60～80 ℃ that filtrate is concentrated into relative density) clear cream, add ethanol to being 70% containing alcohol amount, stir evenly, leave standstill 24 hours, filter, decompression filtrate recycling ethanol is also concentrated into appropriate (containing raw medicinal herbs 5g/ml).Get D101 macroporous absorbent resin (internal diameter 1cm on 5ml solution, long 10cm), absorption, wash with water to colourless, then use 70% ethanol elution to colourless, collect 70% ethanol eluate, evaporate to dryness in water-bath, residue dissolves with 70% methyl alcohol and is settled in 25ml volumetric flask, shakes up, and crosses the miillpore filter of 0.45 μ m as the need testing solution of fresh fish raw meat grass aerial part.Respectively according to 9 need testing solutions of the each preparation of above-mentioned requirements.
4. determination method
Accurate reference substance solution and the each 10 μ l of each need testing solution of drawing respectively, injection liquid chromatography, UV-detector detects, and mensuration wavelength is 254nm, records chromatogram.Calculate the collection of illustrative plates of 9 need testing solutions and the similarity of reference fingerprint, record is as following table.
The reappearance test that table 3 fresh fish raw meat grass aerial part is measured
Detailed mensuration situation and the determination data of (five) 13 batches of fresh cordate houttuynia samples (aerial part) finger-print
1. chromatographic condition
Chromatographic column: Inertsil ODS-3 150 × 4.6mm, 5 μ m
Column temperature: 35 ℃
Flow velocity: 1.0ml/min
Detect wavelength: 254nm
Mobile phase: take 0.2% glacial acetic acid solution as mobile phase A, acetonitrile is Mobile phase B, carries out gradient elution by following program: since 0 minute to 15 minutes, mobile phase A became 89% from 95%, since 15 minutes to 45 minutes, mobile phase A becomes 87% from 89%, and since 45 minutes to 60 minutes, mobile phase A became 65% from 87%, since 60 minutes to 63 minutes, mobile phase A becomes 95% from 65%, and maintains 10 minutes, and gradient is linear gradient.
2. reference substance solution preparation
Get quercitin reference substance appropriate, accurately weighed, add methyl alcohol and make every 1ml containing the solution of 0.1633mg, as with reference to product solution.
Get respectively Hyperoside, chlorogenic acid reference substance is appropriate, accurately weighed, add methyl alcohol and make every 1ml containing Hyperoside 0.0881mg, containing the solution of chlorogenic acid 0.1157mg, product solution in contrast.
3. need testing solution preparation
Get the aerial part of fresh cordate houttuynia, twice of boiling, each 0.5 hour, collecting decoction, filtered, it is 1.18～1.20(60～80 ℃ that filtrate is concentrated into relative density) clear cream, add ethanol to being 70% containing alcohol amount, stir evenly, leave standstill 24 hours, filter, decompression filtrate recycling ethanol is also concentrated into appropriate (containing raw medicinal herbs 5g/ml).Get D101 macroporous absorbent resin (internal diameter 1cm on 5ml solution, long 10cm), absorption, wash with water to colourless, then use 70% ethanol elution to colourless, collect 70% ethanol eluate, evaporate to dryness in water-bath, residue dissolves with 70% methyl alcohol and is settled in 25ml volumetric flask, shakes up, and crosses the miillpore filter of 0.45 μ m as the need testing solution of fresh fish raw meat grass aerial part.The aerial part of fresh cordate houttuynia has following 13 batches, and 13 batch samples all meet relevant regulations of " Chinese Pharmacopoeia " version in 2010, and details see the following form.
Table 4 fresh fish raw meat grass aerial part is collected situation
4. determination method
Accurate each reference substance solution and the each 10 μ l of need testing solution of drawing respectively, injection liquid chromatography, UV-detector detects, and mensuration wavelength is 254nm, records chromatogram.
5. discussion of results:
The relatively finger-print of 13 batches of cordate houttuynia sample aerial parts, finds: a.13 6 of total chromatographic peaks in batch sample; B. by the finger-print of sample 1 ~ 13, draft generation reference fingerprint.The finger-print of other samples and reference fingerprint are compared, in test sample chromatogram, all there are 6 and reference fingerprint characteristic of correspondence peak, pressing similarity evaluation calculates, except S peak, the similarity of test sample finger-print and reference fingerprint is all greater than 0.90.
The similarity data of table 513 batch fresh fish raw meat grass aerial part
6 characteristic peaks corresponding in the standard finger-print of described fresh side cordate houttuynia aerial part are:
Take quercitin as with reference to time, the concrete data at 1-5 peak:
No. 1 peak: relative retention time 0.248, accounts for the Area Ratio 51.0% with reference to peak;
No. 2 peaks: relative retention time 0.333, accounts for the Area Ratio 52.0% with reference to peak;
No. 3 peaks: relative retention time 0.392, accounts for the Area Ratio 24.0% with reference to peak;
No. 4 peaks: relative retention time 0.848, accounts for the Area Ratio 68.0% with reference to peak;
No. 5 peaks: relative retention time 0.867, accounts for the Area Ratio 13.0% with reference to peak;
S peak: for reference to peak (quercitin).
By the characteristic peak of above-mentioned standard finger-print, after calculating by similarity, can draw following tolerance interval:
No. 1 peak: relative retention time 0.224-0.273, accounts for the Area Ratio 26.0%-102.0% with reference to peak;
No. 2 peak: relative retention time 0.300-0.366, account for the Area Ratio 26.0%-105.0% with reference to peak;
No. 3 peak: relative retention time 0.351-0.429, account for the Area Ratio 12.0%-48.0% with reference to peak;
No. 4 peak: relative retention time 0.824-0.870, account for the Area Ratio 34.0%-137.0% with reference to peak;
No. 5 peak: relative retention time 0.843-0.890, account for the Area Ratio 6.0-26.0% with reference to peak;
S peak: for reference to peak.
In sum, the cordate houttuynia overground part extract that the present invention prepares, has contained multiclass effective constituent, and its material base is comparatively clear and definite, can be used as a kind of new standard control thing in quality of medicinal material detection, be more conducive to the quality control to cordate houttuynia aerial part.
The present invention, by the integrated survey to need testing solution preparation method, chromatographic condition, has finally obtained best cordate houttuynia aerial part detection method.While utilizing the inventive method to detect, baseline is steady, each chromatogram peak-to-peak shape and degree of separation are all good, and, flavonoids to cordate houttuynia aerial part, organic acid compound detect simultaneously, from the cordate houttuynia aerial part in 13 batches of different places of production, draw the product general character of this medicinal material---be standard finger-print, for the quality testing of cordate houttuynia aerial part provides reliable method, for the quality of medicinal material of cordate houttuynia aerial part provides guarantee.
Claims (14)
1. a detection method for cordate houttuynia aerial part, is characterized in that: it adopts HPLC finger print method to detect, and comprises following operation steps:
(1) preparation of need testing solution:
Get cordate houttuynia aerial part, supernatant after water extract-alcohol precipitation, remove after ethanol, upper nonpolar or low pole large pore resin absorption column, first washes with water to water lotion colourless, use again 50%～90%V/V ethanol elution, collect ethanol eluate, except after desolventizing, residue dissolves with 50%～90%V/V methyl alcohol, filter, obtain need testing solution;
(2) chromatographic condition:
Chromatographic column: make filling agent with octadecylsilane chemically bonded silica;
Detect wavelength: 254 ± 5nm;
Mobile phase: take 0.1-0.5%V/V glacial acetic acid solution as mobile phase A, acetonitrile is Mobile phase B, carries out gradient elution by following program:
Since 0 minute to 15 minutes, mobile phase A became 89% from 95%, since 15 minutes to 45 minutes, mobile phase A becomes 87% from 89%, and since 45 minutes to 60 minutes, mobile phase A became 65% from 87%, since 60 minutes to 63 minutes, mobile phase A became 95% from 65%;
(3) need testing solution gained chromatogram and standard finger-print are compared, calculate similarity.
2. the detection method of cordate houttuynia aerial part according to claim 1, is characterized in that: in step (1), described macroreticular resin is D101.
3. the detection method of cordate houttuynia aerial part according to claim 1, is characterized in that: in step (1), wash-out is 70%V/V by the concentration of ethanol, and methanol concentration is 70%V/V.
4. the detection method of cordate houttuynia aerial part according to claim 1, it is characterized in that: described water extract-alcohol precipitation refers to: by after the boiling of cordate houttuynia aerial part, the clear cream that when decocting liquid is concentrated into 60～80 ℃, relative density is 1.10～1.30, add ethanol to being 50%～90%V/V containing alcohol amount, leave standstill.
5. the detection method of cordate houttuynia aerial part according to claim 1, is characterized in that: in step (2), described mobile phase A is 0.2%V/V glacial acetic acid solution.
6. the detection method of cordate houttuynia aerial part according to claim 1, is characterized in that: in step (2), detection wavelength is 254nm.
7. the detection method of cordate houttuynia aerial part according to claim 1, is characterized in that: described chromatographic column is Inertsil ODS-3 150 × 4.6mm, 5 μ m.
8. the detection method of cordate houttuynia aerial part according to claim 1, is characterized in that: in step (2), column temperature is 35 ℃, and flow velocity is 1.0ml/min.
9. the detection method of cordate houttuynia aerial part according to claim 1, is characterized in that: in described HPLC finger print method, take a kind of compound in quercitin, Hyperoside, chlorogenic acid or two or more potpourri as reference substance.
10. the detection method of cordate houttuynia aerial part according to claim 1 or 5, is characterized in that: described cordate houttuynia aerial part is saururaceae plant houttuynia cordata H
outtuynia cordatathunb. fresh aerial part.
The detection method of 11. cordate houttuynia aerial parts according to claim 1, it is characterized in that: the feature of described standard finger-print is as follows: have 6 characteristic peaks, take the retention time of quercitin chromatographic peak and peak area as reference, calculate relative retention time and the relative peak area at other peaks, wherein
No. 1 peak: relative retention time is 0.224-0.273, relative peak area is 0.26-1.02;
No. 2 peaks: relative retention time is 0.300-0.366, relative peak area is 0.26-1.05;
No. 3 peaks: relative retention time is 0.351-0.429, relative peak area is 0.12-0.48;
No. 4 peaks: relative retention time is 0.824-0.870, relative peak area is 0.34-1.37;
No. 5 peaks: relative retention time is 0.843-0.890, relative peak area is 0.06-0.26;
The relative retention time of quercitin chromatographic peak is 1, and relative peak area is 1.
The detection method of 12. cordate houttuynia aerial parts according to claim 11, it is characterized in that: the feature of described standard finger-print is as follows: have 6 characteristic peaks, take the retention time of quercitin chromatographic peak and peak area as reference, calculate relative retention time and the relative peak area at other peaks, wherein
No. 1 peak: relative retention time 0.24-0.25, accounts for the Area Ratio 0.4-0.6 with reference to peak;
No. 2 peak: relative retention time 0.33-0.34, account for the Area Ratio 0.4-0.6 with reference to peak;
No. 3 peak: relative retention time 0.39-0.40, account for the Area Ratio 0.2-0.4 with reference to peak;
No. 4 peak: relative retention time 0.84-0.85, account for the Area Ratio 0.6-0.8 with reference to peak;
No. 5 peak: relative retention time 0.86-0.87, account for the Area Ratio 0.1-0.2 with reference to peak;
The relative retention time of quercitin chromatographic peak is 1, and relative peak area is 1.
The detection method of 13. cordate houttuynia aerial parts according to claim 12, it is characterized in that: the feature of described standard finger-print is as follows: have 6 characteristic peaks, take the retention time of quercitin chromatographic peak and peak area as reference, calculate relative retention time and the relative peak area at other peaks, wherein
No. 1 peak: relative retention time 0.248, accounts for the Area Ratio 0.51 with reference to peak;
No. 2 peaks: relative retention time 0.333, accounts for the Area Ratio 0.52 with reference to peak;
No. 3 peaks: relative retention time 0.392, accounts for the Area Ratio 0.24 with reference to peak;
No. 4 peaks: relative retention time 0.848, accounts for the Area Ratio 0.68 with reference to peak;
No. 5 peaks: relative retention time 0.867, accounts for the Area Ratio 0.13 with reference to peak;
The relative retention time of quercitin chromatographic peak is 1, and relative peak area is 1.
The detection method of 14. cordate houttuynia aerial parts according to claim 13, is characterized in that: described standard finger-print as shown in Figure 4.
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| CN107860848A (en) * | 2017-11-15 | 2018-03-30 | 成都中医药大学 | A kind of HPLC methods for detecting cordate houttuynia flavones ingredient content |
| CN108037197B (en) * | 2017-11-27 | 2020-08-07 | 中山市中智药业集团有限公司 | Method for qualitatively and quantitatively analyzing chemical components of non-volatile secondary metabolites of houttuynia cordata wall-broken decoction pieces |
| CN108484388B (en) * | 2018-03-06 | 2021-03-19 | 成都中医药大学 | New compound in houttuynia cordata, preparation method and application thereof |
| CN115372516B (en) * | 2022-08-24 | 2023-11-03 | 浙江惠松制药有限公司 | Method for measuring content of nucleoside components in houttuynia cordata, radix scutellariae and blue mixture intermediate |
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