CN106610406A - Micro-extraction method of honeysuckle - Google Patents

Abstract

The invention discloses a method for extracting and detecting phenolic acid active components in honeysuckle medicinal materials by using β-cyclodextrin as the adsorbent of the matrix solid phase dispersion extraction technology. The method is as follows: take honeysuckle medicinal material powder and β-cyclodextrin adsorbent and add them to a mortar for grinding, take a solid phase extraction column, add a sieve plate at the bottom, fill the column with the ground solid, fill the filler and then top the column Then add the sieve plate, put the filled column on the solid-phase extraction apparatus, take the eluent, inject it into the column, and elute the column, and collect the eluent with a centrifuge tube at the bottom. After the elution, The eluate in the centrifuge tube is vortexed and centrifuged, and the supernatant is taken to be the active ingredient extract of honeysuckle phenolic acids. Compared with the traditional extraction method, the method used in the invention has simpler and quicker steps, saves time and labor, greatly reduces the amount of organic solvents, has remarkable extraction effect, and can effectively extract the phenolic acid components in the honeysuckle.

Description

A kind of micro-extraction method of honeysuckle

technical field

The invention belongs to the field of extraction and detection of natural medicines. It relates to a micro-extraction method of medicinal plants, specifically, it involves the use of β-cyclodextrin as an adsorbent, combined with micro-matrix solid-phase dispersion extraction technology, a new method for extracting the active components of phenolic acids in honeysuckle .

Background technique

Honeysuckle is the dry flower bud or the first flower of Lonicera japonica Thunb. Honeysuckle has heat-clearing and toxic substances removing, the effect of evacuating wind-heat. For carbuncle swollen furunculosis, sore throat, erysipelas, pyretic toxin blood dysentery, wind-heat cold, fever due to febrile disease, etc. Chlorogenic acid (chlorogenic acid), neochlorogenic acid (neochlorogenic acid), isochlorogenic acid (isochlorogenic acid) are the main active ingredients of honeysuckle. Isochlorogenic acid is a mixture, and 3,5-dicaffeoylquinic acid (3,5-O-dicaffeoylquinic acid) and 4,5-dicaffeoylquinic acid (4,5-O-dicaffeoylquinic acid) as its main ingredient. According to reports, chlorogenic acid compounds have pharmacological effects such as antibacterial, choleretic, hemostasis, and increase of white blood cells. And it has the pharmacological effect of significantly enhancing gastrointestinal motility and promoting gastric juice secretion. Chlorogenic acid has the highest content, followed by 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid and neochlorogenic acid.

Traditional Chinese medicine extraction methods include dipping method, percolation method, reflux method, etc. They generally have the disadvantages of large amount of organic solvent, long extraction time, cumbersome operation process, and low extraction rate. Moreover, these methods are easy to lose active ingredients and produce large errors. The use of a large amount of organic solvents has a greater impact on health and has greater pollution to the environment, which does not meet the green chemistry initiative. Solid-phase extraction (Solid-Phase Extraction, SPE) is a sample pretreatment technology developed in recent years, which is mainly used for the separation, purification and concentration of samples. Compared with traditional methods, it can improve the recovery rate of analytes and make analytes It can be effectively separated from interfering components, reducing the sample pretreatment process, and the operation is simple and fast. Widely used in medicine, environment, food, commodity inspection and other fields. However, it also has the disadvantages of large amount of organic solvent, poor repeatability, easy clogging, low flow rate, and small cross-sectional area.

In view of the above limitations, many new extraction methods have been developed on the basis of solid phase extraction, mainly including matrix solid phase dispersion extraction, molecular imprinted solid phase extraction, dispersive liquid phase microextraction, magnetic solid phase extraction, etc. Compared with the traditional solid-phase extraction method, this new type of extraction method has more significant advantages, mainly including less organic solvent consumption, less environmental pollution, simple and fast operation, and high extraction efficiency. Among them, the matrix solid phase dispersion extraction technology is quite representative.

Matrix solid-phase extraction (MSPD, matrix solid-phase dispersion) is a rapid sample processing technology established in 1989 by Professor Barker of Louisiana State University in the United States. The principle is to grind the solid-phase adsorbent with the sample to obtain a semi-dry mixture and pack it into the column as a filler, then rinse the column with different solvents to elute various analytes. It completes the homogenization, extraction, concentration and purification of the sample in one step, avoiding the loss of the analyte caused by the homogenization, extraction and purification of the sample, and at the same time making the pretreatment of the sample easier and improving the analysis speed , reducing the consumption of organic solvents, most of the obtained extracts can be directly analyzed and detected, reducing the operation steps and environmental pollution. In the current literature reports on the matrix solid phase dispersion extraction technology for natural products, the amount of medicinal materials, adsorbents and eluents is relatively large, which not only wastes materials but also causes great pollution to the environment.

A key step in matrix solid-phase dispersive extraction is the selection of adsorbents. At present, the adsorbents used for matrix solid-phase dispersive extraction mainly include silica gel, C18, activated carbon, alumina, florisil, etc.

In 1891, Villier first isolated cyclodextrin from potato starch treated with Bacillus amylobacter. Cyclodextrin (Cyclodextrin, CD) is formed by the action of cyclodextrin glucose residue transferase on starch, glycogen, maltooligosaccharides and other glucose polymers. - Cyclic oligosaccharides linked by 1,4-glucosidic bonds. The common ones are cyclodextrin α, β, and γ. Among them, β-cyclodextrin is cheap and easy to obtain, and is the most widely used. It has a special structure and properties of "hydrophobic in the inner cavity and hydrophilic in the outer wall", and has a "truncated cone" shape in appearance, so it can be used in the water phase. It simulates the hydrophobic pocket of enzymes and forms inclusion complexes with a series of organic molecules. It is widely used in the adsorption and extraction of environmental pollutants, the inclusion and solubilization of water-insoluble drug molecules, the design and construction of supramolecular enzyme imitation systems, etc. field. Moreover, β-cyclodextrin has a moderate cavity size, strong inclusion ability, can be absorbed and decomposed in the human body, and is safe and non-toxic to the human body, so it is suitable as an adsorbent for matrix solid phase dispersion extraction.

So far, β-cyclodextrin has been used as an adsorbent in matrix solid-phase dispersion extraction technology, but there is no report in the field of extraction, and this method is a blank for the extraction and detection of honeysuckle.

Contents of the invention

The present invention aims to solve the problems of low selectivity of adsorbents, large amount of medicinal materials and adsorbents, and large consumption of organic solvents existing in the existing matrix solid-phase dispersion extraction technology. A method for solid-phase dispersion extraction of effective components in honeysuckle medicinal materials. β-cyclodextrin is used as an adsorbent to adsorb and combine target molecules in honeysuckle medicinal materials, so that target molecules can be quickly extracted and eluted from medicinal material powders, and the present invention is implemented in a trace system, which greatly reduces the the amount of organic solvent used.

The technical scheme adopted in the present invention is:

A method for extracting active ingredients of phenolic acids in honeysuckle medicinal materials, wherein the effective ingredients of phenolic acids are neochlorogenic acid, chlorogenic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid One or more than two kinds of acid, the method comprises the following steps:

Weigh the honeysuckle sample, put it in a mortar, add cyclodextrin solid-phase extraction filler, mix and grind with the medicinal material sample thoroughly. Take another solid-phase extraction column, add a sieve plate to the bottom, fill the column with ground powder, fill the filler and then add a sieve plate to the top, and place the filled column on the solid-phase extraction apparatus. Carry out elution, collect the eluate with a centrifuge tube at the bottom, centrifuge, take the supernatant to be the active ingredient extract of phenolic acids, and use ultra-high performance liquid chromatography to detect and analyze.

The types of cyclodextrins are: α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, methyl-β-cyclodextrin, dihydroxypropyl-β-cyclodextrin, preferably β-cyclodextrin - Cyclodextrins.

The mass ratio of the honeysuckle powder to the β-cyclodextrin adsorbent is 1:0.25～4, preferably 1:3.

The grinding time is 60-160s, preferably 120s.

The types of eluents include: methanol, 80% methanol water, 90% methanol water, acetonitrile, ethanol, methanol:ethanol 1:1, preferably 80% methanol water.

The specific experimental operation steps after optimization are:

Weigh 25 mg of honeysuckle herb powder and 75 mg of β-cyclodextrin according to the mass ratio of 1:3, put them into a mortar and grind for 120 seconds, take a 1 mL solid-phase extraction cartridge, add a sieve plate to the bottom, and fill the cartridge with the ground powder. After filling the column, add a sieve plate to the top, place the filled column on a solid-phase extraction apparatus, take 0.5mL80% methanol water, and elute the column, and use a 1.5mL centrifuge tube to collect the eluate at the bottom , then put it into a centrifuge, centrifuge at 13000rpm for 5min, take the supernatant, which is the phenolic acid active ingredient extract, put it into a sample bottle, and use ultra-high performance liquid chromatography (UPLC) for sampling and analysis to obtain the liquid extract of the extract. phase chromatogram;

Then use the reference substance of neochlorogenic acid to prepare a series of reference substance solutions with different concentrations, and detect it with an ultra-high performance liquid chromatography under the same conditions as the extract of honeysuckle to obtain the liquid chromatogram of the reference substance of neochlorogenic acid. The concentration of the reference substance is the abscissa, and the peak area of the chromatographic peak of the new chlorogenic acid reference substance is the ordinate to draw the standard curve of the new chlorogenic acid, and the same method is used to make the chlorogenic acid standard curve, 3,5-dicaffeoylquinic acid Standard curve, 4,5-dicaffeoylquinic acid standard curve; according to the peak area of each chromatographic peak in the liquid chromatogram of the extract and the standard curve of each component, the neochlorogenic acid and chlorogenic acid in the extract are calculated. , 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid content can be converted accordingly to obtain the content of the active ingredients extracted in the extract relative to the honeysuckle powder.

The advantages of the present invention are:

This method is the first to use β-cyclodextrin as the adsorbent of matrix solid-phase dispersion extraction to extract and detect Chinese herbal medicines, which is original and has not been reported in the current literature.

The method carries out extraction and detection under the trace system, and compared with the extraction method described in the Chinese Pharmacopoeia, the use of organic solvents is significantly reduced, and the pollution to the environment is reduced. In addition, the use of medicinal materials and adsorbents is also extremely low, which saves resources and improves environmental protection benefits.

The adsorbent cyclodextrin of the present invention has the special structure and properties of "hydrophobic in the inner cavity and hydrophilic in the outer wall", and has a "truncated cone" shape in appearance, so the adsorption and extraction efficiency is high, and β-cyclodextrin The cavity is moderate in size, strong in inclusion ability, can be absorbed and decomposed in the human body, is safe and non-toxic to the human body, does not pollute the environment, and is cheap and easy to obtain. It makes the experimental operation environment safe and reliable, and is in line with the concept of sustainable development and environmental friendliness in today's advocacy of green chemistry.

The method of the present invention has a series of outstanding characteristics: non-toxic to human body, non-polluting environment; safe and reliable operating environment; for the extraction of natural medicines, the present invention also has the advantages of high efficiency, quick and convenient operation steps, and the like.

Description of drawings

Fig. 1 is the process flow diagram of the matrix solid phase dispersion extraction method of the present invention.

Figure 2 is a histogram of the matrix solid-phase dispersion extraction effect for investigating the types of adsorbents. In the figure, 1, 2, 3, 4 represent different active ingredients in honeysuckle, 1: neochlorogenic acid, 2: chlorogenic acid, 3: 3,5-dicaffeoylquinic acid, 4: 4,5-di Caffeoylquinic acid.

Figure 3 is a histogram of the matrix solid phase dispersion extraction effect under different mass ratios between medicinal materials and β-cyclodextrin. In the figure, 1, 2, 3, and 4 respectively represent different active ingredients in honeysuckle, 1: neochlorogenic acid, 2: chlorogenic acid, 3: 3,5-dicaffeoylquinic acid, 4: 4,5- Dicaffeoylquinic acid. The abscissa in the figure represents the mass of β-cyclodextrin in mg.

Figure 4 is a histogram of the solid phase dispersion extraction effect of the matrix to investigate the type of eluent. In the figure, 1, 2, 3, and 4 respectively represent different active ingredients in honeysuckle, 1: neochlorogenic acid, 2: chlorogenic acid, 3: 3,5-dicaffeoylquinic acid, 4: 4,5- Dicaffeoylquinic acid.

Fig. 5 is a line chart for investigating the effect of matrix solid phase dispersion extraction with different grinding times. In the figure, 1, 2, 3, and 4 respectively represent different active ingredients in honeysuckle, 1: neochlorogenic acid, 2: chlorogenic acid, 3: 3,5-dicaffeoylquinic acid, 4: 4,5- Dicaffeoylquinic acid. The abscissa in the figure represents the milling time.

Fig. 6 is the liquid chromatogram of honeysuckle mixed reference substance. In the figure, 1, 2, 3, and 4 respectively represent different active ingredients in honeysuckle, 1: neochlorogenic acid, 2: chlorogenic acid, 3: 3,5-dicaffeoylquinic acid, 4: 4,5- Dicaffeoylquinic acid.

Fig. 7 is a liquid chromatogram of the extract solution of active ingredients of honeysuckle phenolic acids. In the figure, 1, 2, 3, and 4 represent the active ingredients in honeysuckle, in order: 1: neochlorogenic acid, 2: chlorogenic acid, 3: 3,5-dicaffeoylquinic acid, 4: 4, 5 - Dicaffeoylquinic acid.

detailed description

The detection method provided by the present invention is described in more detail through the following examples. Because of its wide range of applications, there are many specific implementations. The content of the present invention will be further elaborated below in conjunction with the discussion of several examples.

The specific steps of the preparation method of honeysuckle reference substance solution are: take neochlorogenic acid, chlorogenic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid reference substance amount, accurately weighed, Put it in a brown measuring bottle, add methanol to make a reference solution containing 500 μg of neochlorogenic acid, 500 μg of chlorogenic acid, 500 μg of 3,5-dicaffeoylquinic acid, and 500 μg of 4,5-dicaffeoylquinic acid per 1 mL , that is.

Example 1

Weighed 5 parts of honeysuckle herb powder, 25 mg each, and added them to 5 groups of mortars respectively, and weighed different types of cyclodextrins (α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, methyl-β-cyclodextrin, Cyclodextrin, dihydroxypropyl-β-cyclodextrin) 25mg each, added to 5 sets of mortars and ground with honeysuckle powder for 120s. Take five 1mL solid-phase extraction cartridges, add sieves to the bottom, fill the cartridges with ground powder respectively, add sieves to the top after filling, and place them on the solid-phase extraction apparatus. Take 0.5mL of methanol, inject it into the small column, and elute the small column one by one. The bottoms were collected with five 1.5mL centrifuge tubes, and then placed in a centrifuge, centrifuged at 13000rpm for 5min. The supernatant was taken, which was the extract of phenolic acid active ingredients in Flos Lonicerae, and samples were taken and analyzed by ultra-high performance liquid chromatography (UPLC).

The instrument used in the embodiment is an Agilent ultra-high performance liquid chromatography system (Agilent 1290 Infinity LC, Agilent Technologies, Santa Clara, CA, USA) equipped with a vacuum pump, a binary pump mobile phase system, a constant temperature autosampler, and a constant temperature column oven . Chromatographic column: Agilent Zorbax Extend-C18 column (2.1mm×50mm, 1.8μm), detection wavelength: 330nm. Column temperature: 40°C. Injection volume: 1 μL. Flow rate 0.4mL/min, mobile phase: A: 0.1v% formic acid in water, B: methanol. Gradient elution: 0~2min, 0%~30%B; 2~3min, 30%~35%B; 3~4min, 35%~40%B; 4~7min, 40%~50%B; 7~ 8min, 50%～100%B; 8～9min, 100%～100%B; 9～10min, 100%～30%B, equilibration time 5min.

The experimental results are shown in Table 1 below, and the data in Table 1 are peak areas.

Table 1.

1 represents neochlorogenic acid, 2 represents chlorogenic acid, 3 represents 3,5-dicaffeoylquinic acid, and 4 represents 4,5-dicaffeoylquinic acid.

The histogram of the solid phase extraction effect of different kinds of adsorbents is shown in Fig. 2. The results showed that β-cyclodextrin had the best extraction effect. The β-cyclodextrin eluted solution was yellow-green and darkest. It may be due to the moderate molecular hole of β-cyclodextrin that it can better absorb the active ingredients in honeysuckle.

Example 2

Weigh 25 mg each of 9 honeysuckle medicinal powders and add them into 9 groups of mortars respectively, and weigh β-ring Add dextrin to 9 groups of mortars and grind with honeysuckle powder for 120s. Take nine 1mL solid-phase extraction cartridges, add sieve trays to the bottom, fill the cartridges with ground powder respectively, and add sieve trays to the top after filling. Place the filled 9 small columns on the solid phase extraction apparatus. Take 0.5mL of methanol and elute the small columns one by one. The bottoms were collected with nine 1.5mL centrifuge tubes, and then placed in a centrifuge and centrifuged at 13000rpm for 5min. The supernatant was collected as a sample, and analyzed by ultra-high performance liquid chromatography (UPLC).

The experimental results are shown in Table 2 below, and the data in Table 2 are peak areas.

Table 2.

1 represents neochlorogenic acid, 2 represents chlorogenic acid, 3 represents 3,5-dicaffeoylquinic acid, and 4 represents 4,5-dicaffeoylquinic acid.

The influence of the mass ratio between different medicinal materials and β-cyclodextrin on the effect of matrix solid phase dispersion extraction is shown in Figure 3. The results showed that the extraction effect increased sequentially with the increase of the amount of β-cyclodextrin, but after increasing to 75 mg, the extraction effect tended to be flat or even decreased, so 75 mg was selected as the best adsorbent amount in subsequent experiments. The possible reason is that with the increase of the amount of adsorbent, the active ingredient in the mixed solid can be better adsorbed, but the total amount of active ingredient that can be adsorbed is limited. After the amount of adsorbent is increased to the optimal value, the extraction effect tends to be flat. It shows that the active ingredient has basically been adsorbed by the adsorbent. Moreover, too much adsorbent will also make it difficult to desorb the active ingredient from the adsorbent, which will lead to a downward trend in the extraction effect.

Example 3

Weighed 6 parts of tangerine peel medicinal powder, 25 mg each, and added them to 6 groups of mortars, and weighed 6 parts of 75 mg β-cyclodextrin, respectively, and added them to 6 groups of mortars for different grinding times (60s, 80s, 100s, 60s, 80s, 100s, 120s, 140s, 160s). Take 6 solid-phase extraction columns of 1ml size, add sieve plates to the bottom, fill the columns with ground powder respectively, and add sieve plates to the top after filling. Place the filled 6 small columns on the solid phase extraction apparatus. Take 0.5ml of methanol, inject it into the small column, and elute the small column one by one. The bottom was collected with six 1.5ml centrifuge tubes, placed in an ionizer, and centrifuged at 13000rpm for 5min. The supernatant was collected as a sample, and analyzed by ultra-high performance liquid chromatography (UPLC).

The experimental results are shown in Table 3 below, and the data in Table 3 are peak areas.

table 3.

1 represents neochlorogenic acid, 2 represents chlorogenic acid, 3 represents 3,5-dicaffeoylquinic acid, and 4 represents 4,5-dicaffeoylquinic acid.

The histogram of matrix solid phase dispersion extraction effect at different grinding times is shown in Figure 4. The results showed that with the increase of grinding time, the extraction effect gradually increased, and after reaching the optimal value of 120s, the extraction effect tended to decline with the increase of grinding time. The possible reason is that sufficient grinding will help the sorbent to more fully contact the active components in the mixed solids, thereby increasing the extraction efficiency. However, excessive grinding will force the active ingredient to be squeezed into the small and dense pore size of the adsorbent, making the elution difficult and reducing the extraction effect.

Example 4

Weigh 6 parts of honeysuckle medicinal powder, 25 mg each, into 6 groups of mortars, and weigh 6 parts of 75 mg β-cyclodextrin, add them into 6 groups of mortars and grind with honeysuckle medicinal powder for 120 s. Take 6 1mL solid-phase extraction cartridges, add sieve trays to the bottom, fill the cartridges with ground powder respectively, and add sieve trays to the top after filling. Place the filled 6 small columns on the solid phase extraction apparatus. Take 0.5mL of different eluents (methanol, 80% methanol, 90% methanol, ethanol, acetonitrile, methanol:ethanol ratio 1:1), inject them into small columns respectively, and elute the small columns one by one. The bottom was collected with six 1.5mL centrifuge tubes respectively. After the elution is completed, put it into a centrifuge and centrifuge at 13000rpm for 5min. The supernatant was collected as a sample, and analyzed by ultra-high performance liquid chromatography (UPLC).

The experimental results are shown in Table 4 below, and the data in Table 4 are peak areas.

Table 4.

1 represents neochlorogenic acid, 2 represents chlorogenic acid, 3 represents 3,5-dicaffeoylquinic acid, and 4 represents 4,5-dicaffeoylquinic acid.

The histogram of matrix solid phase dispersion extraction effect of different eluents is shown in Figure 5. The results show that the methanol elution effect is better than other eluents, and it is possible that methanol can interact with the target molecule to generate hydrogen bonds. In contrast, the experimental results of the acetonitrile group are the worst among all the investigation groups. The possible reason is that acetonitrile has the weakest ability to dissolve target molecules and interact with the adsorbent. The best extraction with 80% methanol may be due to the closest polarity to the target molecule.

Intraday precision

Weigh 25 mg of honeysuckle powder and 75 mg of β-cyclodextrin, add them into a mortar and grind for 120 seconds. Take a 1mL solid-phase extraction column, add a sieve plate to the bottom, fill the column with ground powder, and add a sieve plate to the top after filling. Place the filled column on the solid phase extraction apparatus. Take 0.5mL of 80% methanol water, inject it into the small column, and elute the small column. Collect the bottom with a 1.5mL centrifuge tube. After the elution is completed, put it into a centrifuge and centrifuge at 13000rpm for 5min. The supernatant was collected as a sample, and analyzed by ultra-high performance liquid chromatography (UPLC). Six consecutive injections were made on the same day.

day-to-day precision

Weigh 25 mg of honeysuckle powder and 75 mg of β-cyclodextrin, add them into a mortar and grind for 120 seconds. Take a 1mL solid-phase extraction column, add a sieve plate to the bottom, fill the column with ground powder, and add a sieve plate to the top after filling. Place the filled column on the solid phase extraction apparatus. Take 0.5mL of 80% methanol water, inject it into the small column, and elute the small column. Collect the bottom with a 1.5mL centrifuge tube. After the elution is completed, put it into a centrifuge and centrifuge at 13000rpm for 5min. The supernatant was collected as a sample, and analyzed by ultra-high performance liquid chromatography (UPLC). This sample was injected twice a day for 3 consecutive days.

The results of intra-day and inter-day precision experiments are summarized in Table 5 below:

table 5

1 represents neochlorogenic acid, 2 represents chlorogenic acid, 3 represents 3,5-dicaffeoylquinic acid, and 4 represents 4,5-dicaffeoylquinic acid.

Repeated inspection

Refer to the following experimental procedures, and do 3 groups in parallel as a repeatability investigation.

Weigh 25 mg of honeysuckle powder and 75 mg of β-cyclodextrin, add them into a mortar and grind for 120 seconds. Take a 1mL solid-phase extraction column, add a sieve plate to the bottom, fill the column with ground powder, and add a sieve plate to the top after filling. Place the filled column on the solid phase extraction apparatus. Take 0.5mL of 80% methanol water, inject it into the small column, and elute the small column. Collect the bottom with a 1.5mL centrifuge tube. After the elution is completed, put it into a centrifuge and centrifuge at 13000rpm for 5min. The supernatant was collected as a sample, and analyzed by ultra-high performance liquid chromatography (UPLC).

Determination of medicinal material content

Weigh 25 mg of honeysuckle powder and 75 mg of β-cyclodextrin, add them into a mortar and grind for 120 seconds. Take a 1mL solid-phase extraction column, add a sieve plate to the bottom, fill the column with ground powder, and add a sieve plate to the top after filling. Place the filled column on the solid phase extraction apparatus. Take 0.5mL of 80% methanol water, inject it into the small column, and elute the small column. Collect the bottom with a 1.5mL centrifuge tube. After the elution is completed, put it into a centrifuge and centrifuge at 13000rpm for 5min. The supernatant was taken, which was the extract of phenolic acid active ingredients, and was sampled and analyzed by ultra-high performance liquid chromatography (UPLC). Obtain a liquid chromatogram of the extract.

Fig. 7 is a liquid chromatogram of the extract solution of active ingredients of phenolic acids. In the figure, 1, 2, 3, and 4 represent different active ingredients in honeysuckle, respectively: 1: neochlorogenic acid, 2: chlorogenic acid, 3: 3,5-dicaffeoylquinic acid, 4: 4 , 5-dicaffeoylquinic acid.

Mixed reference solutions of different concentrations were prepared with reference substances of neochlorogenic acid, chlorogenic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid, and were tested by ultra-high performance liquid chromatography under the same conditions. Chromatographic detection, obtain the chromatograms of the four reference substances, take the injection volume of the four reference substances as the abscissa, and take the peak area of the chromatographic peak in the chromatogram of the four reference substance solutions as the ordinate, respectively make new chlorogenic acid standards curve, chlorogenic acid standard curve, 3,5-dicaffeoylquinic acid standard curve and 4,5-dicaffeoylquinic acid standard curve. The contents of neochlorogenic acid, chlorogenic acid, 3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid in honeysuckle were calculated according to the liquid chromatogram of the extract and the standard curve.

Neochlorogenic acid, chlorogenic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid are the liquid chromatograms of the Flos Lonicerae reference solution of 100 μg/mL concentration as shown in Figure 6 , in the figure, 1, 2, 3, and 4 represent different active ingredients in honeysuckle, respectively: 1: neochlorogenic acid, 2: chlorogenic acid, 3: 3,5-dicaffeoylquinic acid, 4: 4,5-dicaffeoylquinic acid.

The standard curves and limits of detection and limits of quantification of the four components are shown in Table 6 below:

Table 6.

Recovery experiment

Weigh 25 mg of honeysuckle medicinal material powder and 75 mg of β-cyclodextrin, and make 2 groups in parallel. One group adds 50 μL of 100 μg/mL honeysuckle mixed reference substance, and the other group does not add honeysuckle mixed reference substance. Both groups are added to the mortar Grind for 120s. Take two 1mL solid-phase extraction cartridges respectively, add sieve trays to the bottom, fill the cartridges with ground powder, and add sieve trays to the top after filling. Place the filled column on the solid phase extraction apparatus. Take 0.5mL of 80% methanol water and inject them into small columns respectively, and elute the small columns one by one. The bottom was collected with a 1.5mL centrifuge tube. After the elution is completed, put it into a centrifuge and centrifuge at 13000rpm for 5min. The supernatant was collected as a sample, and analyzed by ultra-high performance liquid chromatography (UPLC).

The experimental results of repeatability, content determination and recovery rate are summarized in Table 7 below:

Table 7

The results show that the method of the invention has good repeatability, high recovery rate and high detection accuracy.

Comparative example:

According to the extraction method in the Chinese Pharmacopoeia: about 0.5g of the powder of this product (passed through No. 4 sieve), accurately weighed, put in a stoppered conical flask, accurately add 50% methanol 50ml, weighed, and ultrasonically treated (power 250W, frequency 35kHz) for 30 minutes, let cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, accurately measure 5ml of filtrate, put it in a 25ml brown measuring bottle, add 50% methanol To the scale, shake well, that is.

Gained extract is sampled and analyzed with ultra-high performance liquid chromatography (UPLC), and the liquid chromatogram is compared with the standard curve. It can be measured that the active ingredients in the extract are neochlorogenic acid 2.692mg/g, green Ortho acid 25.05mg/g, 3,5-dicaffeoylquinic acid 16.918mg/g, 4,5-dicaffeoylquinic acid 2.3375mg/g. And the content of active ingredient in the extract liquid that the present application method makes is neochlorogenic acid 3.3719mg/g, chlorogenic acid 33.3368mg/g, 3,5-dicaffeoylquinic acid 20.733mg/g, 4,5-dicaffeoylquinic acid is 2.7641 mg/g, and the extraction effect is significantly improved compared with the pharmacopoeia method.

Claims (6)

1. a kind of Trace bio-element method of natural product, for extracting Chinese medicine honeysuckle in liposoluble ingredient, the phenolic acids Effective ingredient is one or two in neochlorogenic acid, chlorogenic acid, 3,5-diCQA, Isochlorogenic acid C More than, the method comprising the steps of：

(1) Chinese medicine honeysuckle powder and adsorbent are weighed with the mass ratio of certain medical material and adsorbent, adds in mortar and grind Regular hour.1mL specification solid phase extraction columns are taken, bottom adds sieve plate, with ground solid-filling pillar, filler filled out Top adds sieve plate after reality.The pillar filled in is placed on solid-phase extracting instrument.The eluent of appropriate volume is taken, pillar is injected It is interior, the air pump of solid-phase extracting instrument is opened, eluting is carried out to pillar.Collected with 1.5mL specifications centrifuge tube bottom.Eluting terminates Afterwards, by liquid vortex 10s in centrifuge tube, after be put in centrifuge be centrifuged.Supernatant dress sample is taken, Ultra Performance Liquid Chromatography instrument is used (UPLC) sampling analyses, obtain the liquid chromatogram of phenolic acid effective constituents in extracting solution；

(2) reference substance solution of variable concentrations is prepared with the reference substance of neochlorogenic acid, according to extracting solution similarity condition ultra high efficiency Chromatograph of liquid detection, obtains the liquid chromatogram of neochlorogenic acid reference substance, with the sample size of neochlorogenic acid reference substance as horizontal seat Mark, the peak area of chromatographic peak makes neochlorogenic acid standard song as vertical coordinate with the liquid chromatogram of neochlorogenic acid reference substance solution Line, makes in the same way chlorogenic acid standard curve, 3,5-diCQA standard curve, Isochlorogenic acid C Standard curve；According to the peak area and the standard curve of each composition of chromatographic peak in the liquid chromatogram of each composition of extracting solution, meter Calculation obtains the content of neochlorogenic acid in extracting solution, chlorogenic acid, 3,5-diCQA, Isochlorogenic acid C.

2. the method for claim 1, it is characterised in that in the step (1), the adsorbent is beta-schardinger dextrin-.

3. the method for claim 1, it is characterised in that in the step (1), certain medical material and adsorbent Mass ratio is 1: 3.

4. the method for claim 1, it is characterised in that in the step (1), milling time is 120s.

5. the method for claim 1, it is characterised in that in the step (1), the eluent is 80% methanol.

6. the method for claim 1, it is characterised in that in the step (1), the volume of the eluent is 0.5mL.