CN103257187A - Ultra-high pressure extraction of chlorogenic acid and luteoloside in honeysuckle flower and liquid phase analysis method - Google Patents
Ultra-high pressure extraction of chlorogenic acid and luteoloside in honeysuckle flower and liquid phase analysis method Download PDFInfo
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- PZIRUHCJZBGLDY-UHFFFAOYSA-N Caffeoylquinic acid Natural products CC(CCC(=O)C(C)C1C(=O)CC2C3CC(O)C4CC(O)CCC4(C)C3CCC12C)C(=O)O PZIRUHCJZBGLDY-UHFFFAOYSA-N 0.000 title claims abstract description 51
- CWVRJTMFETXNAD-KLZCAUPSSA-N Neochlorogenin-saeure Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)C(=O)O CWVRJTMFETXNAD-KLZCAUPSSA-N 0.000 title claims abstract description 51
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- 229940074393 chlorogenic acid Drugs 0.000 title claims abstract description 51
- FFQSDFBBSXGVKF-KHSQJDLVSA-N chlorogenic acid Natural products O[C@@H]1C[C@](O)(C[C@@H](CC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)C(=O)O FFQSDFBBSXGVKF-KHSQJDLVSA-N 0.000 title claims abstract description 51
- 235000001368 chlorogenic acid Nutrition 0.000 title claims abstract description 51
- BMRSEYFENKXDIS-KLZCAUPSSA-N cis-3-O-p-coumaroylquinic acid Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)cc2)[C@@H]1O)C(=O)O BMRSEYFENKXDIS-KLZCAUPSSA-N 0.000 title claims abstract description 51
- PEFNSGRTCBGNAN-QNDFHXLGSA-N luteolin 7-O-beta-D-glucoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC(O)=C2C(=O)C=C(C=3C=C(O)C(O)=CC=3)OC2=C1 PEFNSGRTCBGNAN-QNDFHXLGSA-N 0.000 title claims abstract description 51
- 241000205585 Aquilegia canadensis Species 0.000 title claims abstract 11
- WJHSRFQBVYHKKL-UHFFFAOYSA-N Oroboside Natural products OC1C(O)C(O)C(CO)OC1OC1=CC(O)=C2C(=O)C(C=3C=C(O)C(O)=CC=3)=COC2=C1 WJHSRFQBVYHKKL-UHFFFAOYSA-N 0.000 title abstract 4
- 238000004458 analytical method Methods 0.000 title description 3
- 239000007791 liquid phase Substances 0.000 title description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 37
- 238000000034 method Methods 0.000 claims abstract description 33
- 239000000284 extract Substances 0.000 claims abstract description 27
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 26
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 18
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims abstract description 13
- 239000002904 solvent Substances 0.000 claims abstract description 13
- 239000006228 supernatant Substances 0.000 claims abstract description 11
- 239000012153 distilled water Substances 0.000 claims abstract description 10
- 239000007788 liquid Substances 0.000 claims abstract description 10
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- 238000004445 quantitative analysis Methods 0.000 claims abstract description 8
- 238000005516 engineering process Methods 0.000 claims abstract description 7
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- 238000002156 mixing Methods 0.000 claims abstract description 4
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- KBGKQZVCLWKUDQ-UHFFFAOYSA-N luteolin-glucoside Natural products OC1C(O)C(O)C(CO)OC1OC1=CC(O)=CC2=C1C(=O)C=C(C=1C=C(O)C(O)=CC=1)O2 KBGKQZVCLWKUDQ-UHFFFAOYSA-N 0.000 claims description 47
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- Extraction Or Liquid Replacement (AREA)
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Abstract
The invention discloses a method for quickly and efficiently extracting chlorogenic acid and luteoloside from honeysuckle and carrying out HPLC quantitative analysis, which comprises the following steps: ultrahigh pressure extraction: firstly, crushing dried honeysuckle and sieving the crushed honeysuckle with a 60-80-mesh sieve, adding water or an organic solvent of ethanol, methanol and n-butyl alcohol into the honeysuckle powder according to a solid-to-liquid ratio of 1: 10-1: 100, mixing and sealing the mixture, soaking the mixture at normal temperature, putting the mixture into a high-pressure container, applying pressure at 20-60 ℃ to leach the mixture for 1-5 min, and centrifugally separating the extract after pressure relief to obtain a honeysuckle extract; quantitative analysis: taking 2ml of the supernatant obtained after centrifugation, putting the supernatant into a 10ml volumetric flask, fixing the volume with distilled water, and refrigerating for later use. The method for extracting chlorogenic acid and luteoloside from honeysuckle flower by using the ultrahigh pressure technology and measuring the content of chlorogenic acid and luteoloside has the characteristics of high extraction rate, short extraction time, small solvent consumption, simplicity and accuracy in operation, good repeatability, no pollution, safety, low energy consumption and the like.
Description
Technical field
Biological technical field of the present invention, be specifically related to a kind of fast, chlorogenic acid and galuteolin and carry out the HPLC quantitative analysis method in the high efficiency extraction honeysuckle.
Background technology
Honeysuckle is the dry flower of caprifoliaceae plant honeysuckle (Lonicera japonica Thunb.) or the flower that band is just opened, having the function of clearing heat and detoxicating, anti-inflammation, hepatic cholagogic, the cool heat wind, antiviral and strengthen the effect of immunity that looses, is one of conventional Chinese medicine material.Chlorogenic acid and galuteolin are representational effective components in the traditional Chinese medicine honeysuckle, and 2010 editions " Chinese pharmacopoeia has increased galuteolin content detection project.The extracting method of chlorogenic acid and galuteolin mainly contains decocting cooking method, organic solvent reflux extraction, ultrasonic extraction, Microwave Extraction method, supercritical CO 2 extraction etc. in the honeysuckle.Decocting cooking method and organic solvent reflux extraction carry out under higher temperature, cause active loss of active ingredients easily, and decompose, change, impurity content height in the product, extraction time is long, and technology is loaded down with trivial details etc.Though and Microwave Extraction and ultrasonic extraction extraction time, energy consumption etc. reduce greatly, exist noise big, be difficult to deficiency such as large-scale production.The supercritical CO 2 extraction exists equipment investment big, expends height, and the material strong to relative molecular mass height, polarity extracts problems such as difficulty.In addition, 2010 editions " Chinese pharmacopoeia adopts different HPLC analytical approachs that the content of chlorogenic acid and galuteolin is measured respectively, and minute is long, is unfavorable for the needs of express-analysis and batch samples quality control.Therefore, develop a kind of fast, chlorogenic acid and galuteolin and simultaneously to these two kinds of methods that composition carries out the HPLC quantitative test in the high efficiency extraction honeysuckle, the quality control of traditional Chinese medicine honeysuckle and Related product is had higher using value.
At present existing superhigh pressure technique is extracted the report of honeysuckle, yet there are no report but use the UHV (ultra-high voltage) extractive technique to extract chlorogenic acid and galuteolin in the honeysuckle and set up the method that a kind of HPLC method measures these two kinds of compositions simultaneously.
Summary of the invention
For overcoming deficiency of the prior art, the invention provides a kind of fast, chlorogenic acid and galuteolin and carry out the HPLC quantitative analysis method in the high efficiency extraction honeysuckle.
For realizing above-mentioned technical purpose, reach above-mentioned technique effect, the present invention is achieved through the following technical solutions:
A kind of fast, chlorogenic acid and galuteolin and carry out the HPLC quantitative analysis method in the high efficiency extraction honeysuckle, comprise the steps:
1) UHV (ultra-high voltage) is extracted: at first with drying honeysuckle pulverized the 60-80 mesh sieve, again Honeysuckle Flower is added water by the solid-to-liquid ratio of 1:10-1:100 or ethanol (concentration is 10%-90%), methyl alcohol, normal butyl alcohol organic solvent, soak 0-24 h at normal temperatures after mixing sealing, again this potpourri is placed high pressure vessel, applying 100-500MPa pressure under 20-60 ℃ leaches, its dwell time is 1-5min, after the release extract is carried out centrifuging, gets Flos Lonicerae extractive solution.
2) quantitative test: get the supernatant 2ml that obtains after centrifugal in the volumetric flask of 10ml, use the distilled water constant volume, refrigerate standby; The HPLC condition is: Cosmosil π-NAP C
18Chromatographic column (5 μ m, 4.6 mm * 250 mm); Phase flows: A:B=acetonitrile: 0.4% phosphoric acid solution; Condition of gradient elution: 0～15min, B:90-80%, 15～30min, B:80%, 30～40min, B:80-70%; Column temperature: 30 ℃; Flow velocity: 1ml/min; Detect wavelength: 327nm, 350nm; Sample size: 20 μ L.
Further, UHV (ultra-high voltage) in the described step (1) is extracted (ultrahigh-pressure extraction, UHPE), full name is " isostatic cool pressing technology ", refer to use at normal temperatures the hydrostatic pressure of 100-1000MPa on the mixed liquor that extracts solvent and Chinese medicine, and under predetermined pressure, keep a period of time, make the vegetable cell external and internal pressure reach release rapidly after the balance.Because the inside and outside seepage pressure of cell increases suddenly, the structure of cell membrane changes, and makes intracellular effective constituent can pass the various films of cell and transfer in the extracellular extract, reaches the purpose of pharmaceutically active ingredient in the extraction.
Compared with prior art, the present invention has following beneficial effect:
(1) traditional extracting method needs heating at present, causes destruction, the sex change of effective constituent in the honeysuckle easily.This method is carried out under 40 ℃, has avoided the effective constituent structural change loss that causes because of thermal effect and the reduction of physiologically active.And, existing research report, biomaterial is subjected to high pressure to do the time spent, have only macromolecular substances generation sex change, and small-molecule substance can be not influenced.Chlorogenic acid and galuteolin can not take place when therefore, UHV (ultra-high voltage) is extracted brokenly change, sex change.
(2) use superhigh pressure technique chlorogenic acid extracting and galuteolin and can shorten extraction time greatly, only be 4 min, it is 2% of refluxing extraction, energy consumes low, UHV (ultra-high voltage) extraction simultaneously is what to carry out in an airtight environment, do not have the volatilization of solvent, so this technology meets requirements of green environmental protection more.
(3) compare with classic method, the efficient height that UHV (ultra-high voltage) is extracted, and also the impurity content in the extract is low, is conducive to separation and the purifying of active substance.
(4) newly-established HPLC analytical approach can be analyzed chlorogenic acid and galuteolin simultaneously, simple, accurately, fast, high, the good reproducibility of tight ness rating, the quality control of traditional Chinese medicine honeysuckle and Related product is had higher using value.
Above-mentioned explanation only is the general introduction of technical solution of the present invention, for can clearer understanding technological means of the present invention, and can be implemented according to the content of instructions, below with preferred embodiment of the present invention describe in detail as after.The specific embodiment of the present invention is provided in detail by following examples.
Embodiment
Below in conjunction with embodiment, describe the present invention in detail.
A kind of fast, chlorogenic acid and galuteolin and carry out the HPLC quantitative analysis method in the high efficiency extraction honeysuckle, comprise the steps:
1) UHV (ultra-high voltage) is extracted: at first with drying honeysuckle pulverized the 60-80 mesh sieve, again Honeysuckle Flower is added water by the solid-to-liquid ratio of 1:10-1:100 or ethanol (concentration is 10%-90%), methyl alcohol, normal butyl alcohol organic solvent, soak 0-24 h at normal temperatures after mixing sealing, again this potpourri is placed high pressure vessel, applying 100-500MPa pressure under 20-60 ℃ leaches, its dwell time is 1-5min, after the release extract is carried out centrifuging, gets Flos Lonicerae extractive solution.
2) quantitative test: get the supernatant 2ml that obtains after centrifugal in the volumetric flask of 10ml, use the distilled water constant volume, refrigerate standby; The HPLC condition is: Cosmosil π-NAP C
18Chromatographic column (5 μ m, 4.6 mm * 250 mm); Phase flows: A:B=acetonitrile: 0.4% phosphoric acid solution; Condition of gradient elution: 0～15min, B:90-80%, 15～30min, B:80%, 30～40min, B:80-70%; Column temperature: 30 ℃; Flow velocity: 1ml/min; Detect wavelength: 327nm, 350nm; Sample size: 20 μ L.
Further, UHV (ultra-high voltage) in the described step (1) is extracted (ultrahigh-pressure extraction, UHPE), full name is " isostatic cool pressing technology ", refer to use at normal temperatures the hydrostatic pressure of 100-1000MPa on the mixed liquor that extracts solvent and Chinese medicine, and under predetermined pressure, keep a period of time, make the vegetable cell external and internal pressure reach release rapidly after the balance.Because the inside and outside seepage pressure of cell increases suddenly, the structure of cell membrane changes, and makes intracellular effective constituent can pass the various films of cell and transfer in the extracellular extract, reaches the purpose of pharmaceutically active ingredient in the extraction.
Embodiment 1: the different solvents experiment
With drying honeysuckle pulverized the 60-80 mesh sieve, accurately the Honeysuckle Flower of weighing 1.0g drying is put in the plastic closures bag for 4 parts, adds the water of 50ml, 70% ethanol, methyl alcohol, normal butyl alcohol respectively, sealing was soaked 24 hours.Put into extra-high tension unit then, 30 ℃ of temperature are extracted in pressurization 300MPa leaching, dwell time 5min, and release is got supernatant 2ml in the volumetric flask of 10ml after centrifugal, use the distilled water constant volume, and employing HPLC method is measured the content of chlorogenic acid and galuteolin.The HPLC condition is: Cosmosil π-NAP C
18Chromatographic column (5 μ m, 4.6 mm * 250 mm); Phase flows: A:B=acetonitrile: 0.4% phosphoric acid solution; Condition of gradient elution: 0～15min, B:90-80%, 15～30min, B:80%, 30～40min, B:80-70%; Column temperature: 30 ℃; Flow velocity: 1ml/min; Detect wavelength: 327nm, 350nm; Sample size: 20 μ L.The extraction ratio that calculates chlorogenic acid and galuteolin sees Table 1.
Table 1: the different solvents extract yield relatively
| Solvent | Chlorogenic acid (%) | Galuteolin (%) |
| Normal butyl alcohol | 0.223 | 0.016 |
| Methyl alcohol | 3.486 | 0.083 |
| Water | 4.350 | 0.022 |
| 70% ethanol | 4.357 | 0.073 |
As shown in Table 1,70% second alcohol and water is all very high to the extraction ratio of chlorogenic acid, is respectively 4.357% and 4.350%, but water is very low to the extraction ratio of galuteolin, only is 0.022%.Though methyl alcohol to 0.083%, only be 3.486% to the extraction ratio of chlorogenic acid, and the toxicity of methyl alcohol is far longer than ethanol to the extraction rate reached of galuteolin.Take all factors into consideration security and extraction ratio, we select ethanol as extracting solvent.
Embodiment 2: the solvent strength test
With drying honeysuckle pulverized the 60-80 mesh sieve, accurately the Honeysuckle Flower of weighing 1.0g drying is put in the plastic closures bag for 5 parts, adds 10%, 30%, 50%, 70%, 90% the ethanol of 50ml respectively, sealing was soaked 24 hours.Put into extra-high tension unit then, 30 ℃ of temperature are extracted in pressurization 300MPa leaching, dwell time 5min, and release is got supernatant 2ml in the volumetric flask of 10ml after centrifugal, use the distilled water constant volume, and employing HPLC method is measured the content of chlorogenic acid and galuteolin.The HPLC condition is: Cosmosil π-NAP C
18Chromatographic column (5 μ m, 4.6 mm * 250 mm); Phase flows: A:B=acetonitrile: 0.4% phosphoric acid solution; Condition of gradient elution: 0～15min, B:90-80%, 15～30min, B:80%, 30～40min, B:80-70%; Column temperature: 30 ℃; Flow velocity: 1ml/min; Detect wavelength: 327nm, 350nm; Sample size: 20 μ L.The extraction ratio that calculates chlorogenic acid and galuteolin sees Table 2.
Table 2: different solvents concentration extract yield relatively
| Concentration of alcohol (%) | Chlorogenic acid (%) | Galuteolin (%) |
| 10 | 4.145 | 0.029 |
| 30 | 4.269 | 0.046 |
| 50 | 4.348 | 0.069 |
| 70 | 4.357 | 0.073 |
| 90 | 1.360 | 0.043 |
Different concentrations of alcohol is different to the extraction ratio of chlorogenic acid and galuteolin, selects suitable solvent strength, can improve yield.As known from Table 2, when concentration of ethanol increased, the yield of chlorogenic acid and galuteolin increased.And when concentration of alcohol was 90%, the yield of chlorogenic acid and galuteolin obviously reduced.Therefore, the optimum concentration range of ethanol is 10-70%.
Embodiment 3: the comparison test of different extraction pressure
With drying honeysuckle pulverized the 60-80 mesh sieve, accurately the Honeysuckle Flower of weighing 1.0g drying is put in the plastic closures bag for 5 parts, adds 70% ethanol of 50ml respectively, sealing was soaked 24 hours.Put into extra-high tension unit then, pressurize 100,200,300,400 respectively, the 500MPa leaching, extract 30 ℃ of temperature, dwell time 5min, release, get supernatant 2ml after centrifugal in the volumetric flask of 10ml, use the distilled water constant volume, adopt the HPLC method to measure the content of chlorogenic acid and galuteolin.The HPLC condition is: Cosmosil π-NAP C
18Chromatographic column (5 μ m, 4.6 mm * 250 mm); Phase flows: A:B=acetonitrile: 0.4% phosphoric acid solution; Condition of gradient elution: 0～15min, B:90-80%, 15～30min, B:80%, 30～40min, B:80-70%; Column temperature: 30 ℃; Flow velocity: 1ml/min; Detect wavelength: 327nm, 350nm; Sample size: 20 μ L.The extraction ratio that calculates chlorogenic acid and galuteolin sees Table 3.
Table 3: the different pressure extract yields that extract compare
| Pressure (MPa) | Chlorogenic acid (%) | Galuteolin (%) |
| 100 | 4.052 | 0.062 |
| 200 | 4.041 | 0.069 |
| 300 | 4.188 | 0.084 |
| 400 | 4.406 | 0.082 |
| 500 | 4.414 | 0.078 |
From experimental result as can be seen, when extracting pressure at 100 ~ 400MPa, the extraction ratio of chlorogenic acid and galuteolin increases along with the increase of pressure, when pressure surpasses 400MPa, the extraction ratio of chlorogenic acid decreases, take all factors into consideration extraction ratio and equipment cost, we select 400MPa to extract pressure.
Embodiment 4: different extraction time comparison tests
With drying honeysuckle pulverized the 60-80 mesh sieve, accurately the Honeysuckle Flower of weighing 1.0g drying is put in the plastic closures bag for 5 parts, adds 70% ethanol of 50ml respectively, sealing was soaked 24 hours.Put into extra-high tension unit then, 30 ℃ of temperature are extracted in pressurization 400MPa leaching, respectively pressurize 1,2,3,4,5min, release is got supernatant 2ml in the volumetric flask of 10ml after centrifugal, use the distilled water constant volume, adopt the HPLC method to measure the content of chlorogenic acid and galuteolin.The HPLC condition is: Cosmosil π-NAP C
18Chromatographic column (5 μ m, 4.6 mm * 250 mm); Phase flows: A:B=acetonitrile: 0.4% phosphoric acid solution; Condition of gradient elution: 0～15min, B:90-80%, 15～30min, B:80%, 30～40min, B:80-70%; Column temperature: 30 ℃; Flow velocity: 1ml/min; Detect wavelength: 327nm, 350nm; Sample size: 20 μ L.The extraction ratio that calculates chlorogenic acid and galuteolin sees Table 4.
Table 4: different dwell time extract yields relatively
| Dwell time (min) | Chlorogenic acid (%) | Galuteolin (%) |
| 1 | 4.093 | 0.067 |
| 2 | 4.186 | 0.068 |
| 3 | 4.140 | 0.068 |
| 4 | 4.175 | 0.068 |
| 5 | 3.824 | 0.062 |
From experimental result, the dwell time, effective constituent stripping basically was complete in 2min, and the extraction ratio basically identical of pressurize 4min and pressurize 2min, but in order to guarantee that extracting temperature can reach setting value, we select 4min is extraction time.
Embodiment 5: the comparison test of different extraction temperature
With drying honeysuckle pulverized the 60-80 mesh sieve, accurately the Honeysuckle Flower of weighing 1.0g drying is put in the plastic closures bag for 5 parts, adds 70% ethanol of 50ml respectively, sealing was soaked 24 hours.Put into extra-high tension unit then, pressurization 400MPa leaching is extracted temperature and is respectively 20,30,40,50,60 ℃, pressurize 4min, release is got supernatant 2ml in the volumetric flask of 10ml after centrifugal, use the distilled water constant volume, adopt the HPLC method to measure the content of chlorogenic acid and galuteolin.The HPLC condition is: Cosmosil π-NAP C
18Chromatographic column (5 μ m, 4.6 mm * 250 mm); Phase flows: A:B=acetonitrile: 0.4% phosphoric acid solution; Condition of gradient elution: 0～15min, B:90-80%, 15～30min, B:80%, 30～40min, B:80-70%; Column temperature: 30 ℃; Flow velocity: 1ml/min; Detect wavelength: 327nm, 350nm; Sample size: 20 μ L.The extraction ratio that calculates chlorogenic acid and galuteolin sees Table 5.
Table 5: the different temperature extract yields that extract compare
| The extraction temperature (℃) | Chlorogenic acid (%) | Galuteolin (%) |
| 20 | 1.658 | 0.040 |
| 30 | 1.545 | 0.049 |
| 40 | 4.168 | 0.064 |
| 50 | 4.027 | 0.063 |
| 60 | 3.748 | 0.055 |
As known from Table 5, when extracting temperature at 20 ~ 40 ℃, the extraction ratio of chlorogenic acid and galuteolin increases along with the raising of temperature, when pressure surpasses 40 ℃, the extraction ratio of chlorogenic acid and galuteolin decreases, and the too high thermally sensitive composition that may destroy in the honeysuckle of temperature, taking all factors into consideration, we select 40 ℃ for extracting temperature.
Embodiment 6: different solid is test frequently
With drying honeysuckle pulverized the 60-80 mesh sieve, accurately the Honeysuckle Flower of weighing 1.0g drying is put in the plastic closures bag for 5 parts, adds 10,25,50,75 respectively, 70% ethanol of 100ml, sealing was soaked 24 hours.Put into extra-high tension unit then, pressurization 400MPa leaching, extracting temperature is 40 ℃, pressurize 4min, release is got supernatant 2ml in the volumetric flask of 10ml after centrifugal, use the distilled water constant volume, and employing HPLC method is measured the content of chlorogenic acid and galuteolin.The HPLC condition is: Cosmosil π-NAP C
18Chromatographic column (5 μ m, 4.6 mm * 250 mm); Phase flows: A:B=acetonitrile: 0.4% phosphoric acid solution; Condition of gradient elution: 0～15min, B:90-80%, 15～30min, B:80%, 30～40min, B:80-70%; Column temperature: 30 ℃; Flow velocity: 1ml/min; Detect wavelength: 327nm, 350nm; Sample size: 20 μ L.The extraction ratio that calculates chlorogenic acid and galuteolin sees Table 6.
Table 6: different solid than extract yield relatively
| Solid-to-liquid ratio (g/ml) | Chlorogenic acid (%) | Galuteolin (%) |
| 1:10 | 3.863 | 0.073 |
| 1:25 | 4.168 | 0.076 |
| 1:50 | 4.395 | 0.076 |
| 1:75 | 4.521 | 0.072 |
| 1:100 | 4.580 | 0.070 |
Work as solid-to-liquid ratio as can be seen from the above table in 1:10 ~ 1:100 scope, along with the increase of solid-to-liquid ratio, the extraction ratio of chlorogenic acid and galuteolin increases gradually.But consider post-processing difficulty and economy that extracts active ingredients is separated.Therefore, the optimum range of solid-to-liquid ratio is 1:10 ~ 1:75.
The selection principle of UHV (ultra-high voltage) extraction process parameter is: guaranteeing product quality, improving under the prerequisite of yield, selecting lower pressure, short extraction time, lower solvent strength, less solid-to-liquid ratio as far as possible, to reduce cost of equipment, energy resource consumption.Therefore our chlorogenic acid of obtaining and the optimal processing parameter of galuteolin are: 50% ethanol, extract pressure 300MPa, dwell times 4 min, extract 40 ℃ of temperature, solid-to-liquid ratio 1:25.Under these process conditions, the yield of chlorogenic acid and galuteolin is respectively 4.971% and 0.089%.
Embodiment 7: the Different Extraction Method comparison test
Extract that the method for chlorogenic acid and galuteolin has multiple, different extracting method in the honeysuckle, yield is different with extraction process.For this reason, we with same batch of sample, compare the result with superhigh pressure extracting method and other extracting method
See Table 7.
Table 7: the Different Extraction Method extract yield relatively
From experimental result as can be known, compare with refluxing extraction, the chlorogenic acid yield that UHV (ultra-high voltage) is extracted has improved 43%, and galuteolin is constant substantially, but extraction time is 2% of refluxing extraction, so the UHV (ultra-high voltage) extraction has the yield height, advantages such as extraction time is short, energy consumption is low, environmental protection.
The above is the preferred embodiments of the present invention only, is not limited to the present invention, and for a person skilled in the art, the present invention can have various changes and variation.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (2)
- One kind fast, chlorogenic acid and galuteolin and carry out the HPLC quantitative analysis method in the high efficiency extraction honeysuckle, comprise the steps:1) UHV (ultra-high voltage) is extracted: at first with drying honeysuckle pulverized the 60-80 mesh sieve, again Honeysuckle Flower is added water by the solid-to-liquid ratio of 1:10-1:100 or ethanol (concentration is 10%-90%), methyl alcohol, normal butyl alcohol organic solvent, soak 0-24 h at normal temperatures after mixing sealing, again this potpourri is placed high pressure vessel, apply 100-500MPa pressure and leach under 20-60 ℃, its dwell time is 1-5min, after the release extract is carried out centrifuging, get Flos Lonicerae extractive solution2) quantitative test: get the supernatant 2ml that obtains after centrifugal in the volumetric flask of 10ml, use the distilled water constant volume, refrigerate standby; The HPLC condition is: Cosmosil π-NAP C 18Chromatographic column (5 μ m, 4.6 mm * 250 mm); Phase flows: A:B=acetonitrile: 0.4% phosphoric acid solution; Condition of gradient elution: 0～15min, B:90-80%, 15～30min, B:80%, 30～40min, B:80-70%; Column temperature: 30 ℃; Flow velocity: 1ml/min; Detect wavelength: 327nm, 350nm; Sample size: 20 μ L.
- 2. according to claim 1 quick, chlorogenic acid and galuteolin and carry out the HPLC quantitative analysis method in the high efficiency extraction honeysuckle, it is characterized in that, UHV (ultra-high voltage) in the described step (1) is extracted (ultrahigh-pressure extraction, UHPE), full name is " isostatic cool pressing technology ", refer to use at normal temperatures the hydrostatic pressure of 100-1000MPa on the mixed liquor that extracts solvent and Chinese medicine, and under predetermined pressure, keep a period of time, make the vegetable cell external and internal pressure reach release rapidly after the balance, because the inside and outside seepage pressure of cell increases suddenly, the structure of cell membrane changes, make intracellular effective constituent can pass the various films of cell and transfer in the extracellular extract, reach the purpose of pharmaceutically active ingredient in the extraction.
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| CN104288204A (en) * | 2014-05-27 | 2015-01-21 | 河南科技大学 | Method for extracting chlorogenic acid from honeysuckle flower |
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