CN105399786A - Extraction method of iridoid glycosides in Lamiophlomis rotata - Google Patents

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

Extraction method of iridoid glycosides in Duyiwei

technical field

The invention relates to an extraction method of iridoid glycosides in Duyiwei, in particular to a microwave extraction method.

Background technique

Unique flavor is the dry aerial part of Lamiophlomisrotata (Benth) Kudo, the unique flavor of Lamiophlomis rotata (Benth) Kudo. It has the function of promoting blood circulation to stop bleeding, expelling wind and relieving pain, and drying yellow water, which is used to treat bruises, traumatic bleeding, rheumatic arthralgia, yellow water disease and other diseases. Existing studies have shown that iridoid glycosides are one of the main active ingredients of Duyiwei, and have strong hemostatic and coagulation-promoting effects. Therefore, it is of great significance to extract as many iridoid glycosides in Duyiwei as possible.

At present, for the extraction method of the active ingredients in Duyiwei, the content of total flavonoids is mainly used as the extraction index, and there is no report on the iridoid glycosides in Duyiwei. At the same time, for the iridoid glycosides in Duyiwei, the focus of the existing reports is how to better separate and purify the iridoid glycosides from the water extract of Duyiwei, for example, "Duyiwei Determination of total iridoid glycosides, total flavonoids and total phenylethanol glycosides in different extracts" (Qiu Jianguo et al., Chinese Journal of Experimental Formulas, 2013, Issue 23, pages 119-124), "Industrial Extraction, Separation and Purification "Total iridoid glycosides components of Yiwei" (Qiu Jianguo et al., Chinese Journal of Experimental Formulas, Issue 13, 2011, pages 32-35), etc., did not pay attention to how to extract more iridoid ethers from Yiwei Terpene glycosides.

Therefore, there is an urgent need for an extraction method for iridoid glycosides in Duyiwei.

Contents of the invention

In order to solve the above problems, the present invention provides a method for extracting iridoid glycosides in Duyiwei, comprising the following steps:

Take the unique medicinal material, pulverize it, add an alcoholic solvent with a concentration of 10%-70%, and extract it by microwave. The volume-mass ratio of the alcoholic solvent to the medicinal material is 20-40:1mL/g. Preferably, the alcoholic solvent is selected from methanol, ethanol or n-butanol, most preferably ethanol.

Preferably, the alcohol solvent has a concentration of 10%-22%; more preferably, the alcohol solvent has a concentration of 20%-22%, most preferably 22%.

Preferably, the volume-to-mass ratio of the alcoholic solvent to the medicinal material is 35-36:1 mL/g, most preferably 36:1 mL/g.

Preferably, the temperature of the microwave extraction is 40°C-80°C; more preferably, the temperature of the microwave extraction is 60°C-70°C, most preferably 63°C.

Preferably, the microwave extraction takes 6-10 minutes, most preferably 8 minutes.

Preferably, the concentration of the alcoholic solvent is 20-22%, the volume-to-mass ratio of the alcoholic solvent to the medicinal material is 35-36:1mL/g, and the temperature of microwave extraction is 60-63°C; more preferably, the ethanol The concentration is 22%, the volume-mass ratio of ethanol to medicinal material is 36:1mL/g, and the temperature of microwave extraction is 63°C.

Preferably, the unique herbal medicine is the leaves of the unique herbal medicine; more preferably, the unique herbal medicine is the leaves of the unique herbal medicine dried at 60°C.

The present invention also provides a unique extract, which is prepared by the aforementioned extraction method.

The present invention uses alcohol solvent as the extraction solvent for the first time and combines it with microwave-assisted extraction technology for the extraction of iridoid glycosides in Duyiwei. It has low energy consumption, simple and quick operation, short extraction time and high extraction rate, and is suitable for popularization application.

At the same time, the inventors found in experiments that when water is used to extract the iridoid glycosides in Duyiwei, the extraction effect is not good. The reason for the analysis is that iridoid glycosides are more sensitive to acid, and its glycosidic bonds are easily hydrolyzed by acid. Therefore, when carrying out water extraction of iridoid glycosides, it is necessary to mix calcium carbonate or barium hydroxide into the medicinal materials to prevent the influence of enzymes and organic acids. However, in the present invention, the iridoid glycosides in Duyiwei can be efficiently extracted without adding other reagents to protect the iridoid glycosides by combining alcohol solvents with microwave-assisted extraction, which greatly simplifies the extraction method.

In addition, the 2010 edition of the "Chinese Pharmacopoeia" made a major change to the medicinal parts of Duyiwei, changing from using the whole plant to the aerial part to protect its wild resources. The inventors found in their research (Zhong Shihong, Gu Rui, Wang Lingxin, etc. HPLC simultaneous determination of the contents of 7 ingredients in Duyiwei, Chinese Journal of Traditional Chinese Medicine, 2014, 39(22): 4373-4378) that there is Good correlation, the average value of the iridoid glycoside components shanzhiside methyl ester and 8-O-acetyl shanzhiside methyl ester in the above-ground part samples is 1.44%, and the above-ground part has more components than the underground part , the content is higher, and it is more reasonable to use the aerial part as medicine. Therefore, the method for effectively extracting iridoid glycosides of the present invention also provides an effective guarantee for further improving the unique quality standard.

Apparently, according to the above content of the present invention, according to common technical knowledge and conventional means in this field, without departing from the above basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above-mentioned content of the present invention will be further described in detail below through specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Description of drawings

Fig. 1 is the chromatogram of the reference substance, in which: 1-caterin; 2-shanzhiside methyl ester; 3-8-O-acetylshanzhiside methyl ester.

Fig. 2 is the spectrogram of cataside.

Fig. 3 Spectrogram of shanzhiside methyl ester.

Fig. 4 is the spectrogram of 8-O-acetylshanzhiside methyl ester.

Fig. 5 is a typical sample chromatogram 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 a spectrogram of chromatographic peak 4 in Fig. 5 .

Fig. 10 is the spectrogram of chromatographic peak 5 in Fig. 5.

Fig. 11 is the spectrogram of chromatographic peak 6 in Fig. 5.

Fig. 12 is the spectrogram of chromatographic peak 7 in Fig. 5.

Figure 13 shows the results of extraction solvent screening tests.

Figure 14 shows the results of the extraction temperature screening test.

Figure 15 shows the results of the ethanol concentration screening test.

Figure 16 is the result of the liquid-to-solid ratio screening test.

Figure 17 shows the results of extraction time screening experiments.

detailed description

The source of material and instrument used in the present invention is as follows:

The unique medicinal material (purchased in Aba County, Sichuan Province in July 2013) was identified as the dried leaves of the unique Lamiophlomisrotata (Benth) Kudo; chromatographically pure methanol, acetonitrile, and other reagents were analytically pure, and the reagents were all from Chengdu Kelong Chemical Reagent Factory .

UltiMate3000 HPLC, Diana, Germany; Discover microwave synthesizer, CEM, USA; JE series multifunctional electronic balance, Shanghai Puchun Measuring Instrument Co., Ltd.; Chinese herbal medicine pulverizer, Tianjin Qinsite Instrument Co., Ltd.; electric blast Drying oven, Shanghai Yiheng Scientific Instrument Co., Ltd.

In the present invention, the content of total iridoid glycosides in the unique medicinal material powder is detected by high performance liquid chromatography, and the extraction is compared by comparing the total peak area of the total iridoid glycosides in the extract. Effect.

The high performance liquid chromatography conditions are: WelchromC18 chromatographic column, acetonitrile-0.1% phosphoric acid aqueous solution gradient elution system. The mobile phase is A: acetonitrile-B: 0.1% phosphoric acid aqueous solution, gradient elution program: 0~15min, 8%~12%A; 15~20min, 12%~19%A; 20~35min, 19%~31% a. Volume flow: 1.0mL/min, detection wavelength: 238nm, column temperature: 30°C.

Identify the iridoid glycoside chromatographic peaks in the spectrum with the spectrogram, and add up their total peak areas as the basis for determination.

Fig. 1 is the chromatogram of the reference substances of catalin, shanzhiside methyl ester and 8-O-acetyl shanzhiside methyl ester.

Since the iridoid glycosides in Duyiwei all have similar spectra, according to the comparison of the spectrum of the known reference substance in Figure 2-4 and the spectrum of 1-7 chromatographic peaks in the sample of Figure 5, it is judged to find Figure 5 Seven main iridoid glycoside peaks in the sample spectrum.

The identification results are as follows: chromatographic peaks 1-7 are all iridoid glycosides, and the basis for screening conditions in this paper is the total peak area, which refers to the sum of the areas of these 7 chromatographic peaks, and the 3rd, 4th, and 7th chromatographic peaks are respectively related to Chromatographic peaks No. 1, 2, and 3 of the reference substance correspond to chromatographic peaks, that is, chromatographic peaks No. 3, 4, and 7 are catarin, shanzhiside methyl ester, and 8-O-acetylshanzhiside methyl ester, respectively.

Therefore, this method is accurate and reliable for the detection of iridoid glycosides in Duyiwei, with good repeatability and stable results.

Embodiment 1 extraction method of the present invention

1. Material preparation

Duyiwei leaves are dried in a blast drying oven at 60°C; coarse powder is made, and the powder is sealed and stored in a dark and dry place for later use.

2. Extraction

Weigh 1.000g of Duyiwei medicinal material powder into a 100mL round bottom flask, add 36.0mL of 22% ethanol solution, and extract by microwave at 63°C for 8min.

3. Determination

After making up the weight loss of the extract, seal it and let it stand for 5 minutes, pour the supernatant into a 10mL centrifuge tube, seal it and let it stand for 30 minutes, use a 5mL clean syringe to absorb an appropriate amount of supernatant, filter it with a 0.45um filter head, and take the filtrate and inject it into the Appropriate amount in the sample bottle, high performance liquid chromatography sample injection determination.

After repeating three times, the total peak areas of iridoid glycosides were measured to be 711.099×10 ³ , 725.4306×10 ³ , and 721.3918×10 ³ respectively. The calculated RSD value of the values measured in the three experiments was 1.03%, which proves that the method of the present invention can efficiently The iridoid glycosides in Duyiwei are extracted, and the method has high stability and reliable data.

The single factor screening test of embodiment 2 extraction method of the present invention

The screening test of extraction method of the present invention is carried out in the following manner:

Duyiwei leaves are dried in a blast drying oven at 60°C; coarse powder is made, and the powder is sealed and stored in a dark and dry place for later use.

Weigh a certain amount of Duyiwei medicinal material powder into a 100mL round bottom flask, use a certain concentration and volume of ethanol, a certain time, and a certain temperature to perform microwave extraction on the unique medicinal material powder. After making up the weight loss of the extract, seal it and let it stand for 5 minutes, pour the supernatant into a 10mL centrifuge tube, seal it and let it stand for 30 minutes, use a 5mL clean syringe to absorb an appropriate amount of supernatant, filter it with a 0.45um filter head, and take the filtrate and inject it into the Appropriate amount in the sample bottle for high performance liquid chromatography injection.

1. Screening of extraction solvent

In this experiment, the most commonly used solvent was used for microwave extraction of iridoid glycosides in Duyiwei; 3 samples were weighed, each 0.500g, and ultrapure water, 50% methanol solution, and 40.0% each of 50% ethanol solution were added precisely. mL, microwave extraction was carried out at 60°C for 8 minutes, and the results are shown in Figure 13.

The results show that the water extraction effect is not good. The reason for the analysis is that iridoid glycosides are sensitive to acid, and its glycosidic bonds are easily hydrolyzed by acid. Therefore, when carrying out water extraction of iridoid glycosides, it is often necessary to mix in Calcium carbonate or barium hydroxide to protect against enzymes and organic acids.

At the same time, both 50% methanol and 50% ethanol can effectively extract the iridoid glycosides in Duyiwei, and the total peak areas of the two iridoid glycosides are not much different. Considering the safety of the microwave experiment, ethanol was selected as the Extraction solvent for this screening experiment.

After the solvent is determined to be ethanol, the effects of extraction temperature, ethanol concentration, liquid-solid ratio and time on the extraction effect are investigated respectively, and the influence of each factor on the extraction effect is compared.

2. Selection of extraction temperature (°C)

Weigh 5 parts of Duyiwei medicinal material powder, each 0.500g, each precisely add 40.0mL of 20% ethanol solution, under the condition of microwave extraction for 8min, respectively investigate the temperature of 40°C, 50°C, 60°C, 70°C, 80°C Extraction amount, the result is shown in Figure 14.

The results showed that the iridoid glycosides in Duyiwei could be effectively extracted at 40°C-80°C. Among them, the extraction effect is better at 60-70°C.

It can be seen that within the temperature range investigated, the total peak area of iridoid glycosides is almost proportional to the temperature in the increasing stage, reaching the highest at 60°C, and then decreasing slowly.

3. Screening of ethanol concentration (%)

Weigh 7 parts of Duyiwei medicinal material powder, each 0.500g, add 10%, 20%, 30%, 40%, 50%, 60%, 70% ethanol solution 40mL, set the microwave extraction temperature to 60°C, and the extraction time 8min, investigate the influence of ethanol concentration on the extraction efficiency, the results are shown in Figure 15.

The results show that when the concentration of ethanol is 10-70%, the iridoid glycosides in Duyiwei can be effectively extracted. Wherein, the extraction effect is better when the concentration of ethanol is 10-20%.

It can be seen that when the ethanol concentration is less than 20%, the extraction amount of iridoid glycosides in Duyiwei is relatively high, and tends to be gentle. With the increase of ethanol concentration, the extraction amount of iridoid glycosides declines more obviously. Multiple experimental verifications have confirmed that high concentration of ethanol is not effective for microwave extraction of iridoid glycosides in Duyiwei.

4. Screening of liquid to material ratio (mL/g)

Weigh 5 parts of Duyiwei medicinal material powder, each 1.000g, set the microwave extraction temperature to 60°C, the extraction time to 8min, use 20% ethanol solution as the solvent, and investigate the ratio of liquid to material: 20:1, 25:1, 30: 1, 35:1, 40:1 under each condition to the extraction quantity efficiency of iridoid glycosides, the result is shown in Figure 16.

The results showed that when the volume/mass ratio of ethanol to medicinal material was 20-40mL/g, the iridoid glycosides in Duyiwei could be effectively extracted. Among them, the extraction effect is better when the volume-mass ratio of ethanol to medicinal material is 35mL/g.

It can be seen that with the increase of the liquid-to-solid ratio, the extraction amount of iridoid glycosides in Duyiwei has been showing a steady upward trend, reaching the highest at 35:1mL/g, and showing a sharp increase after exceeding 35:1mL/g. downward trend.

5. Selection of extraction time (min)

Weigh 5 parts of Duyiwei medicinal material powder, each 0.500g, add 40.0mL of 20% ethanol liquid precisely, and examine the extraction time under the condition of microwave extraction temperature of 60°C for 4min, 6min, 8min, 10min, and 12min respectively. The results of the extraction amount of iridoid glycosides are shown in Figure 17.

The results show that when the microwave extraction time is 6-10 minutes, the iridoid glycosides in Duyiwei can be effectively extracted. Among them, the extraction effect is better at 8 minutes.

It can be seen that the extraction amount of iridoid glycosides increases first with the increase of time, but as the extraction time continues to increase, the extraction amount decreases slowly.

The process screening of embodiment 3 extraction method of the present invention

According to the method of Example 2, further screening tests were carried out on the extraction method of the present invention with three factors of ethanol concentration, temperature and liquid-to-material ratio.

Among them, the dosage of Duyiwei powder is 1.000g, and the microwave extraction time is 8min.

According to the result of embodiment 2 single-factor screening, the screening range of ethanol concentration is 10-30%, the screening range of extraction temperature is 50-70 ℃, the screening range of liquid-solid ratio is 30-40g/mL, the results are shown in Table 1 Show:

The multifactor screening of table 1 extraction method of the present invention

Experiment number Ethanol concentration (%) Extraction temperature (℃) Liquid to solid 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 twenty two 63 36 719.307**

*5 measurement results: the average value of 707.986, 722.7451, 694.45, 685.317, and 723.072.

**Three determination results: average of 711.099, 725.4306, 721.3918.

The results show that when the concentration of ethanol is 20-22%, the volume-to-mass ratio of ethanol to medicinal material is 35-36:1mL/g, and the temperature of microwave extraction is 60-63°C, the extraction effect is significantly better than other experimental conditions. Among them, when the concentration of ethanol is 22%, the volume-mass ratio of ethanol to medicinal material is 36:1mL/g, and the temperature of microwave extraction is 63°C, the extraction effect is the best.

In summary, the present invention uses alcohol solvents as extraction solvents for the first time, combined with microwave-assisted extraction technology, for the extraction of iridoid glycosides in Duyiwei, with low energy consumption, simple and quick operation, short extraction time, and high extraction rate High, suitable for popularization and application.

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.