CN105399786A - Extraction method of iridoid glycosides in Lamiophlomis rotata - Google Patents
Extraction method of iridoid glycosides in Lamiophlomis rotata Download PDFInfo
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Abstract
The invention discloses an extraction method of iridoid glycosides in Lamiophlomis rotate. The method comprises the following steps: taking a Lamiophlomis rotata medicinal material, crushing the medicinal material, adding an alcohol solvent with the concentration of 10-70%, and carrying out microwave extraction, wherein a volume/mass ratio of the alcohol solvent to the medicinal material is 20-40:1 mL/g. The method adopts combination of use of the alcohol solvent as an extraction solvent with a microwave assisted extraction technology to extract iridoid glycosides in Lamiophlomis rotate, so the method has the advantages of low energy consumption, simple and fast operation, short extraction time, high extraction rate, and suitableness for popularization and application.
Description
Technical field
The present invention relates to the extracting method of iridoid glycoside in Root of Common Lamiophlomis, particularly relate to a kind of microwave extract method.
Background technology
Root of Common Lamiophlomis is the dry aerial parts of labiate Root of Common Lamiophlomis Lamiophlomisrotata (Benth) Kudo.Have promoting blood circulation and hemostasis, wind-expelling pain-stopping, the function of dry yellow water, is used for the treatment of wound, traumatic hemorrhage, rheumatic arthralgia, the diseases such as grasserie.Now there are some researches show, iridoid glycoside is one of main active ingredient of Root of Common Lamiophlomis, has stronger hemostasis, hemostasis effect.Therefore, the iridoid glycoside constituents extracted in Root of Common Lamiophlomis has great importance as much as possible.
At present, for the extracting method of activeconstituents in Root of Common Lamiophlomis, mainly with the content of total flavones for extract index, do not have the report for iridoid glycoside constituents in Root of Common Lamiophlomis.Simultaneously, for the iridoid glycoside constituents in Root of Common Lamiophlomis, the focus of existing report is also how better by the separation and purification from the aqueous extract of Root of Common Lamiophlomis of iridoid glycoside constituents, such as " total iridoid glycoside in the different extract of Root of Common Lamiophlomis, the mensuration of total flavones and total benzyl carbinol glycosides " (Qiu Jianguo etc., Chinese experimental pharmacology of traditional Chinese medical formulae magazine, 23 phases in 2013, 119-124 page), " industrialization extraction separation and purification Radix Lamiophlomidis Rotatae total iridoid glycosides constituents " (Qiu Jianguo etc., Chinese experimental pharmacology of traditional Chinese medical formulae magazine, 13 phases in 2011, 32-35 page) etc., be not concerned about and how extract more iridoid glycoside from Root of Common Lamiophlomis.
Therefore, a kind of extracting method for iridoid glycoside in Root of Common Lamiophlomis is needed at present badly.
Summary of the invention
For solving the problem, the invention provides the extracting method of iridoid glycoside in Root of Common Lamiophlomis, comprising the following steps:
Get Root of Common Lamiophlomis medicinal material, pulverize, adding concentration is 10%-70% alcoholic solvent, microwave extraction, and described alcoholic solvent is 20-40:1mL/g with the volume mass ratio of medicinal material.Preferably, described alcoholic solvent is selected from methyl alcohol, ethanol or propyl carbinol, most preferred ethanol.
Preferably, the concentration of described alcoholic solvent is 10%-22%; Still more preferably, the concentration of described alcoholic solvent is 20%-22%, most preferably 22%.
Preferably, described alcoholic solvent and the volume mass of medicinal material are than being 35-36:1mL/g, most preferably 36:1mL/g.
Preferably, the temperature of described microwave extraction is 40 DEG C-80 DEG C; More preferably, the temperature of described microwave extraction is 60 DEG C-70 DEG C, most preferably 63 DEG C.
Preferably, the time of described microwave extraction is 6-10 minute, most preferably 8 minutes.
Preferably, the concentration of described alcoholic solvent is 20-22%, and alcoholic solvent is 35-36:1mL/g with the volume mass ratio of medicinal material, and the temperature of microwave extraction is 60-63 DEG C; More preferably, the concentration of ethanol is 22%, and ethanol is 36:1mL/g with the volume mass ratio of medicinal material, and the temperature of microwave extraction is 63 DEG C.
Preferably, described Root of Common Lamiophlomis medicinal material is Root of Common Lamiophlomis leaf portion; More preferably, described Root of Common Lamiophlomis medicinal material is the Root of Common Lamiophlomis leaf portion of drying at 60 DEG C.
Present invention also offers a kind of Radix Lamiophlomidis Rotatae extract, described extract prepares with aforesaid extracting method.
The present invention, first using alcoholic solvent as Extraction solvent, combines with microwave auxiliary extraction technology, and for the extraction of iridoid glycoside in Root of Common Lamiophlomis, energy consumption is low, simple and efficient to handle, extraction time is short, extraction rate is high, is suitable for applying.
Meanwhile, contriver finds in an experiment, and when employing, existing conventional Extraction solvent---when water extracts the iridoid glycoside in Root of Common Lamiophlomis, extraction effect is not good.Analyzing reason is that iridoid glycoside is more responsive to acid, and its glycosidic bond is very easily acid hydrolysis.When therefore water extraction being carried out to iridoid glycoside, calcium carbonate or hydrated barta need be admixed in medicinal material, to prevent enzyme and organic acid impact.And the present invention is by combining alcoholic solvent with microwave auxiliary extraction, now without the need to adding other reagent protection iridoid glycoside, high efficiency extraction can goes out iridoid glycoside in Root of Common Lamiophlomis, significantly simplifying extracting method.
In addition, within 2010, version " Chinese Pharmacopoeia " medicinal part to Root of Common Lamiophlomis has done great change, changes over-ground part into, to protect its wild resource by use herb.Contriver is (Zhong Shihong under study for action, Gu Rui, Wang Lingxin, Deng 7 kinds of component contents in .HPLC Simultaneously test Root of Common Lamiophlomis, CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2014,39 (22): 4373-4378) find, over-ground part respectively becomes between grouping has good dependency, the average of the iridoid glycoside composition mountain Cape jasmine glycosides methyl esters in over-ground part sample and 8-O-acetyl mountain Cape jasmine glycosides methyl esters total amount is 1.44%, over-ground part is more than underground part composition kind, and content is higher, is used as medicine more reasonable with over-ground part.Therefore, the present invention effectively extracts the method for iridoid glycoside, and the quality standard also for improving Root of Common Lamiophlomis further provides effective guarantee.
Obviously, according to foregoing of the present invention, according to ordinary technical knowledge and the customary means of this area, not departing under the present invention's above-mentioned basic fundamental thought prerequisite, the amendment of other various ways, replacement or change can also be made.
The embodiment of form by the following examples, is described in further detail foregoing of the present invention again.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following example.All technology realized based on foregoing of the present invention all belong to scope of the present invention.
Accompanying drawing explanation
Fig. 1 is reference substance color atlas, wherein: 1-flax belongs to glycosides; 2-mountain Cape jasmine glycosides methyl esters; 3-8-O-acetyl mountain Cape jasmine glycosides methyl esters.
Fig. 2 is that flax belongs to glycosides spectrogram.
Fig. 3 mountain Cape jasmine glycosides methyl esters spectrogram.
Fig. 4 is 8-O-acetyl mountain Cape jasmine glycosides methyl esters spectrogram.
Fig. 5 is typical sample color atlas of the present invention.
Fig. 6 is the spectrogram of chromatographic peak 1 in Fig. 5.
Fig. 7 is the spectrogram of chromatographic peak 2 in Fig. 5.
Fig. 8 is the spectrogram of chromatographic peak 3 in Fig. 5.
Fig. 9 is the spectrogram of chromatographic peak 4 in Fig. 5.
Figure 10 is the spectrogram of chromatographic peak 5 in Fig. 5.
Figure 11 is the spectrogram of chromatographic peak 6 in Fig. 5.
Figure 12 is the spectrogram of chromatographic peak 7 in Fig. 5.
Figure 13 is the result of Extraction solvent shaker test.
Figure 14 is the result of Extracting temperature shaker test.
Figure 15 is the result of alcohol concn shaker test.
Figure 16 is the result of liquid ratio shaker test.
Figure 17 is the result of extraction time shaker test.
Embodiment
The source of material therefor of the present invention and instrument is as follows:
Root of Common Lamiophlomis medicinal material (being purchased from Aba County, Sichuan in 2013 7) is the dry leave of Root of Common Lamiophlomis Lamiophlomisrotata (Benth) Kudo through mirror; Chromatographic Pure Methanol, acetonitrile, all the other reagent are analytical pure, and reagent is all from Chengdu Ke Long chemical reagent factory.
UltiMate3000 high performance liquid chromatograph, Germany wears peace; Discover Microwave synthesize instrument, U.S. CE M; JE series multifunctional electronic balance, Shanghai Pu Chun measuring instrument company limited; Crusher for Chinese herbal medicine, Tianjin Qin Site Instrument Ltd.; Electric drying oven with forced convection, Shanghai Yiheng Scientific Instruments Co., Ltd.
In the present invention, the total iridoid glycoside content in Root of Common Lamiophlomis medicinal powder adopts high performance liquid chromatography to detect, and compares extraction effect by the size of the total peak area comparing total iridoid methods of glycosides in extracting solution.
High performance liquid chromatography condition is: WelchromC18 chromatographic column, second eyeball-0.1% phosphate aqueous solution gradient elution system.Moving phase is A: acetonitrile-B:0.1% phosphate aqueous solution, gradient elution program: 0 ~ 15min, 8% ~ 12%A; 15 ~ 20min, 12% ~ 19%A; 20 ~ 35min, 19% ~ 31%A.Volumetric flow rate 1.0mL/min, determined wavelength: 238nm, column temperature: 30 DEG C.
Each iridoid glycosides chromatographic peak spectrogram in collection of illustrative plates is pointed out, and adds with its total peak area as mensuration foundation.
Fig. 1 is the reference substance color atlas that flax belongs to glycosides, mountain Cape jasmine glycosides methyl esters and 8-O-acetyl mountain Cape jasmine glycosides methyl esters.
Because iridoid glycosides in Root of Common Lamiophlomis all has similar spectrogram, contrast according to the spectrogram 6-12 of 1-7 chromatographic peak in the known reference substance spectrogram of Fig. 2-4 and Fig. 5 sample, judge to find out 7 main iridoid glycoside chromatographic peaks in Fig. 5 sample drawing spectrum.
Point out result as follows: 1-7 chromatographic peak is iridoid glycoside, the foundation of screening conditions is total peak area herein, namely this 7 chromatographic peak area sums are referred to, wherein the 3rd, 4, No. 7 chromatographic peak is corresponding with 1,2, No. 3 chromatographic peak of reference substance chromatogram respectively, and namely the 3rd, 4, No. 7 chromatographic peak is respectively flax and belongs to glycosides, mountain Cape jasmine glycosides methyl esters and 8-O-acetyl mountain Cape jasmine glycosides methyl esters.
Therefore, this method is to the composition detection of iridoid glycoside in Root of Common Lamiophlomis accurately and reliably, reproducible, and result is stablized.
Embodiment 1 extracting method of the present invention
1, material preparation
The air dry oven that Root of Common Lamiophlomis leaf is placed in 60 DEG C is dried; Beat meal, powder saves backup in dark dry place's sealing.
2, extract
Take Root of Common Lamiophlomis medicinal powder 1.000g in 100mL round-bottomed flask, add 22% ethanolic soln 36.0mL, microwave extraction 8min at 63 DEG C.
3, measure
Extracting solution is supplied weightless rear sealing and standing 5min, pour its supernatant liquor in 10mL centrifuge tube sealing and standing 30min, appropriate with the clean syringe Aspirate supernatant of 5mL, filter with the filter of 0.45um, get subsequent filtrate and inject sample injection bottle in right amount, high performance liquid chromatography sample introduction measures.
In triplicate, the total peak area recording iridoid glycoside is respectively 711.099 × 10
3, 725.4306 × 10
3, 721.3918 × 10
3, testing the value that records for three times, to calculate RSD value be 1.03%, and prove that the inventive method high efficiency extraction can go out iridoid glycoside in Root of Common Lamiophlomis, and method stability is high, data are reliable.
The single factor test shaker test of embodiment 2 extracting method of the present invention
The shaker test of extracting method of the present invention is carried out in the following manner:
The air dry oven that Root of Common Lamiophlomis leaf is placed in 60 DEG C is dried; Beat meal, powder saves backup in dark dry place's sealing.
Take a certain amount of Root of Common Lamiophlomis medicinal powder in 100mL round-bottomed flask, adopt certain alcohol concn and volume, certain hour, and certain temperature carries out microwave extraction to Root of Common Lamiophlomis medicinal powder.Extracting solution is supplied weightless rear sealing and standing 5min, pour its supernatant liquor in 10mL centrifuge tube sealing and standing 30min, appropriate with the clean syringe Aspirate supernatant of 5mL, filter with the filter of 0.45um, get subsequent filtrate and inject sample injection bottle in right amount, for high performance liquid chromatography sample introduction.
1, the screening of Extraction solvent
This experiment selects the most frequently used solvent to carry out microwave extraction to the iridoid glycoside in Root of Common Lamiophlomis; Take 3 parts, sample, every part of 0.500g, precision adds ultrapure water respectively, 50% methanol solution, and each 40.0mL of 50% ethanolic soln, at 60 DEG C of temperature, carry out the microwave extraction of 8min, result as shown in figure 13.
Result shows, water extraction poor effect, and analyzing reason is that iridoid glycoside is more responsive to acid, and its glycosidic bond is very easily acid hydrolysis, and when therefore carrying out water extraction to iridoid glycoside, often need admix calcium carbonate or hydrated barta in medicinal material, to prevent enzyme and organic acid impact.
Meanwhile, 50% methyl alcohol and 50% ethanol all effectively can propose the iridoid glycoside in Root of Common Lamiophlomis, and the total peak area of both iridoid glycosides is more or less the same, and considers the security of microwave experiment, select ethanol as the Extraction solvent of this screening experiment.
After solvent is defined as ethanol, then investigate Extracting temperature respectively, alcohol concn, liquid ratio and time, more each factor affected size to extraction effect on the impact of extraction effect.
2, the screening of Extracting temperature (DEG C)
Take Root of Common Lamiophlomis medicinal powder 5 parts, each 0.500g, each precision adds 20% ethanolic soln 40.0mL, and under the condition of microwave extraction 8min, investigate 40 DEG C respectively, 50 DEG C, 60 DEG C, 70 DEG C, the extracted amount of 80 DEG C of each temperature, result as shown in figure 14.
Result shows, and at 40 DEG C-80 DEG C, all effectively can extract the iridoid glycoside in Root of Common Lamiophlomis.Wherein, during 60-70 DEG C, extraction effect is better.
Can find out, in investigated temperature range, the total peak area of iridoid glycoside was almost directly proportional to temperature in the increase stage, reaches the highest, slowly reduce on the contrary afterwards in 60 DEG C time.
3, the screening of alcohol concn (%)
Take Root of Common Lamiophlomis medicinal powder 7 parts, each 0.500g, each precision adds 10%, and 20%, 30%, 40%, 50%, 60%, 70% ethanolic soln 40mL, arranging microwave extraction temperature is 60 DEG C, and extraction time is 8min, and investigate alcohol concn to the impact of extraction efficiency, result as shown in figure 15.
Result shows, and the concentration of ethanol, when 10-70%, all effectively can extract the iridoid glycoside in Root of Common Lamiophlomis.Wherein, when the concentration of ethanol is 10-20%, extraction effect is better.
Can find out, when alcohol concn is less than 20%, in Root of Common Lamiophlomis, the extracted amount of iridoid glycoside is higher, and tends towards stability, and along with the increase of alcohol concn, the extracted amount downtrending of iridoid glycoside is more obvious.Many experiments checking confirms, the ethanol of high density is to the iridoid glycoside poor effect in microwave extraction Root of Common Lamiophlomis.
4, the screening of liquid ratio (mL/g)
Take Root of Common Lamiophlomis medicinal powder 5 parts, each 1.000g, arranging microwave extraction temperature is 60 DEG C, and extraction time is 8min, ethanolic soln with 20% is as solvent, and investigating liquid ratio is 20:1,25:1,30:1, to the extracted amount efficiency of iridoid glycoside under each condition of 35:1,40:1, result as shown in figure 16.
Result shows, and when ethanol is 20-40mL/g with the volume mass ratio of medicinal material, all effectively can extract the iridoid glycoside in Root of Common Lamiophlomis.Wherein, ethanol is better than extraction effect when being 35mL/g with the volume mass of medicinal material.
Can find out, along with the increase of liquid ratio, in Root of Common Lamiophlomis, the extracted amount of iridoid glycoside is always in the trend that grows steadily, and reaches the highest when 35:1mL/g, then presents sharply downtrending on the contrary more than after 35:1mL/g.
5, the screening of extraction time (min)
Take Root of Common Lamiophlomis medicinal powder 5 parts, each 0.500g, respectively accurate 20% ethanol liquid adding 40.0mL, be under the condition of 60 DEG C, investigate extraction time 4min, 6min, 8min respectively in microwave extraction temperature, the size of the extracted amount of iridoid glycoside when 10min, 12min, result as shown in figure 17.
Result shows, and the time of microwave extraction, when 6-10 minute, all effectively can extract the iridoid glycoside in Root of Common Lamiophlomis.Wherein, 8 minutes time extraction effect better.
Can find out, the extracted amount of iridoid glycoside first increases along with the increase of time presents, but increases along with the continuation of extraction time, and extracted amount instead slowly reduces.
The craft screening of embodiment 3 extracting method of the present invention
According to the method for embodiment 2, with alcohol concn, temperature and liquid ratio three kinds of factors, further shaker test is carried out to extracting method of the present invention.
Wherein, the consumption of Root of Common Lamiophlomis powder is 1.000g, and the microwave extraction time is 8min.
According to the result of embodiment 2 single factor test screening, the screening scope of alcohol concn is 10-30%, and the screening scope of Extracting temperature is 50-70 DEG C, and the screening scope of liquid ratio is 30-40g/mL, and result is as shown in table 1:
The multifactor screening of table 1 extracting method of the present invention
| Experiment number | Alcohol concn (%) | Extracting temperature (DEG C) | Liquid ratio (mL/g) | Peak area (× 10 3) |
| 1 | 10 | 50 | 35 | 455.53 |
| 2 | 30 | 50 | 35 | 575.5323 |
| 3 | 10 | 70 | 35 | 629.02 |
| 4 | 30 | 70 | 35 | 634.252 |
| 5 | 10 | 60 | 30 | 541.588 |
| 6 | 30 | 60 | 30 | 649.435 |
| 7 | 10 | 60 | 40 | 616.717 |
| 8 | 30 | 60 | 40 | 660.0804 |
| 9 | 20 | 50 | 30 | 479.363 |
| 10 | 20 | 70 | 30 | 613.509 |
| 11 | 20 | 50 | 40 | 544.692 |
| 12 | 20 | 70 | 40 | 687.376 |
| 13 | 20 | 60 | 35 | 706.714* |
| 14 | 22 | 63 | 36 | 719.307** |
* 5 measurement results: 707.986, the average of 722.7451,694.45,685.317,723.072.
* * 3 measurement results: 711.099, the average of 725.4306,721.3918.
Result shows, and when the concentration of ethanol is 20-22%, ethanol is 35-36:1mL/g with the volume mass ratio of medicinal material, and when the temperature of microwave extraction is 60-63 DEG C, extraction effect is significantly better than other test conditionss.Wherein, when the concentration of ethanol is 22%, ethanol is 36:1mL/g with the volume mass ratio of medicinal material, and when the temperature of microwave extraction is 63 DEG C, extraction effect is best.
In sum, the present invention, first using alcoholic solvent as Extraction solvent, combines with microwave auxiliary extraction technology, and for the extraction of iridoid glycoside in Root of Common Lamiophlomis, energy consumption is low, simple and efficient to handle, extraction time is short, extraction rate is high, is suitable for applying.
Claims (10)
1. the extracting method of iridoid glycoside in Root of Common Lamiophlomis, is characterized in that: comprise the following steps:
Get Root of Common Lamiophlomis medicinal material, pulverize, adding concentration is 10%-70% alcoholic solvent, microwave extraction, and described alcoholic solvent is 20-40:1mL/g with the volume mass ratio of medicinal material.
2. extracting method according to claim 1, is characterized in that: the concentration of described alcoholic solvent is 10%-22%, and preferred concentration is 20%-22%
3. extracting method according to claim 1, is characterized in that: described alcoholic solvent is 35-36:1mL/g with the volume mass ratio of medicinal material.
4. extracting method according to claim 1, is characterized in that, the temperature of described microwave extraction is 40 DEG C-80 DEG C.
5. extracting method according to claim 4, is characterized in that: the temperature of described microwave extraction is 60 DEG C-70 DEG C.
6. extracting method according to claim 1, is characterized in that: the time of described microwave extraction is 6-10 minute.
7. extracting method according to claim 1, is characterized in that: the concentration of described alcoholic solvent is 20-22%, and alcoholic solvent is 35-36:1mL/g with the volume mass ratio of medicinal material, and the temperature of microwave extraction is 60-63 DEG C.
8. the extracting method according to any one of claim 1-7, is characterized in that: described alcoholic solvent is selected from methyl alcohol, ethanol or propyl carbinol.
9. the extracting method according to any one of claim 1-8, is characterized in that: described Root of Common Lamiophlomis medicinal material is Root of Common Lamiophlomis leaf portion.
10. a Radix Lamiophlomidis Rotatae extract, is characterized in that: described extract prepares with the extracting method described in any one of claim 1-10.
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