阅读说明：本技术 一种分离提取鬼针聚炔苷和鬼针聚炔苷b的制备工艺 (Preparation process for separating and extracting bidens bipinnata polyacetylene and bidens bipinnata polyacetylene B ) 是由 郝鹏飞 李超 高丽 梁鹏 黄显章 张超云 于 2021-08-23 设计创作，主要内容包括：本发明提供了一种分离提取鬼针聚炔苷和鬼针聚炔苷B的制备工艺,属于炔苷类化合物提取技术领域。其包括：取鬼针草粉末,渗漉,浓缩,萃取；将萃取浸膏配制成乙醇混合溶液,离心,取上清液分次上样于树脂柱,采用乙醇进行梯度洗脱,将第一洗脱液减压回收溶剂得到洗脱浸膏；将洗脱浸膏配制成乙醇混合溶液,离心,取上清液加入到聚酰胺柱,先以适量水冲洗,再以乙醇进行梯度洗脱；将第二洗脱液配制成甲醇混合溶液,分次上样于小孔吸附树脂柱,采用甲醇进行梯度洗脱；将第三洗脱液以葡聚糖凝胶进行分离,以甲醇进行洗脱；将第四洗脱液浓缩,以甲醇-水作为流动相,采用制备高效液相系统进行制备,即得。该工艺可提高鬼针聚炔苷和鬼针聚炔苷B的收率。(The invention provides a preparation process for separating and extracting a bidens parviflora and a bidens parviflora B, belonging to the technical field of alkyne glycoside compound extraction. It includes: percolating herba Bidentis Bipinnatae powder, concentrating, and extracting; preparing the extract into an ethanol mixed solution, centrifuging, taking supernatant, sampling on a resin column in times, performing gradient elution by adopting ethanol, and recovering the solvent from the first eluent under reduced pressure to obtain an eluted extract; preparing the eluted extract into an ethanol mixed solution, centrifuging, taking supernatant, adding the supernatant into a polyamide column, washing with a proper amount of water, and performing gradient elution with ethanol; preparing the second eluent into a methanol mixed solution, loading the methanol mixed solution into a small-pore adsorption resin column in a grading manner, and performing gradient elution by adopting methanol; separating the third eluate with Sephadex, and eluting with methanol; and concentrating the fourth eluent, taking methanol-water as a mobile phase, and preparing by adopting a high performance liquid preparation system to obtain the compound. The process can improve yield of polyglycoside B.)

1. A preparation process for separating and extracting the bidens bipinnata polyacetylene and the bidens bipinnata polyacetylene B is characterized by comprising the following steps: the method comprises the following steps:

step S1: percolating herba Bidentis Bipinnatae powder by soaking in ethanol, concentrating percolate to obtain fluid extract, extracting the fluid extract with solvent, and recovering solvent under reduced pressure to obtain extract;

step S2: preparing the extract into an ethanol mixed solution, centrifuging, taking supernatant, sampling in a macroporous adsorption resin column in batches, performing gradient elution by adopting ethanol, and recovering the solvent from the first eluent after the gradient elution under reduced pressure to obtain an eluted extract;

step S3: preparing the elution extract into an ethanol mixed solution, centrifuging, taking supernatant, adding the supernatant into a polyamide column, washing with a proper amount of water, and performing gradient elution with ethanol to obtain a second eluent;

step S4: preparing the second eluent into a methanol mixed solution, loading the methanol mixed solution into a small-pore adsorption resin column in a grading manner, and performing gradient elution by adopting methanol to obtain a third eluent; separating the third eluent by Sephadex LH-20, and eluting by methanol to obtain a fourth eluent;

step S5: and concentrating the fourth eluent, taking methanol-water as a mobile phase, and preparing by adopting a high performance liquid preparation system.

2. The process for preparing the separation and extraction of the bidens pilosa polyacetylene B as claimed in claim 1, wherein the process comprises the following steps: the percolation is carried out by ethanol soaking, and the method specifically comprises the following steps: placing herba Bidentis Bipinnatae powder in percolation barrel, slowly adding 75-90% ethanol until the powder is completely soaked and the ethanol liquid level is 9-11cm higher than the medicinal material powder, sealing the percolation barrel opening with plastic membrane, and soaking for 24-72 hr; then starting percolation, collecting the percolation liquid at the speed of 5-10 ml/(kg.min), filtering, and supplementing 75-90% ethanol by volume fraction at any time to obtain the total percolation liquid.

3. The process for preparing the separation and extraction of the bidens pilosa polyacetylene B as claimed in claim 2, wherein the process comprises the following steps: the volume of the total percolate is 10-15 times of the weight of the bidens bipinnata in Kg in terms of L.

4. The process for preparing the separation and extraction of the bidens pilosa polyacetylene B as claimed in claim 1, wherein the process comprises the following steps: the solvent in step S1 is petroleum ether, ethyl acetate and n-butanol.

5. The process for preparing the separation and extraction of the bidens pilosa polyacetylene B as claimed in claim 1, wherein the process comprises the following steps: the macroporous adsorption resin column is macroporous adsorption resin column D101, AB-8, HP-20 or XAD-4.

6. The process for preparing the separation and extraction of the bidens pilosa polyacetylene B as claimed in claim 1, wherein the process comprises the following steps: and in the step S2, ethanol is adopted for gradient elution, and the volume concentration of the ethanol is 20-100%.

7. The process for preparing the separation and extraction of the bidens pilosa polyacetylene B as claimed in claim 1, wherein the process comprises the following steps: the volume concentration of the ethanol in the step S3 of gradient elution by ethanol is 5-95%.

8. The process for preparing the separation and extraction of the bidens pilosa polyacetylene B as claimed in claim 1, wherein the process comprises the following steps: in the step S4, the volume concentration of methanol in gradient elution with methanol is 35-65%, and the volume concentration of methanol in elution with methanol is 75-85%.

9. The process for preparing the separation and extraction of the bidens pilosa polyacetylene B as claimed in claim 1, wherein the process comprises the following steps: the volume ratio of methanol to water in the mobile phase of methanol-water is 33: 67.

Technical Field

The invention belongs to the technical field of alkyne glycoside compounds, and particularly relates to a preparation process for separating and extracting bidens pilosa polyacetylene glycoside and bidens pilosa polyacetylene glycoside B.

Background

Bidens pilosa, also known as herba Lasiosphaera Seu Calvatia and Veronica officinalis, belongs to the Compositae, Bidens, and is distributed in south America, tropical and subtropical regions. The folk medicine has a long history, has the effects of clearing heat and removing toxicity, eliminating stasis and reducing swelling and the like, and has wide curative effect and definite curative effect.

Modern research shows that: bidens pilosa contains flavonoids, polyacetylene (glycosides), terpenoids, phenolic acids, ocannins and other compounds, and the pharmacological activity of Bidens pilosa is intensively reflected in aspects such as antimalarial, anti-inflammatory, blood fat reduction, blood pressure reduction, blood sugar reduction, oxidation resistance, tumor resistance and the like, particularly the cardiovascular protection and oxidation resistance are deeply concerned by researchers and accepted by people, and at present, China develops various clinical formulas containing Bidens pilosa plants, so that the research on Bidens pilosa has potential social value and scientific research value.

The polyacetylene component is a main effective component contained in the bidens bipinnata and has good biological activity in the aspects of antimicrobial, cytotoxic and antitumor activities and the like.

The patent document with the publication number of CN106939028A relates to alkyne glycoside compounds and an extraction and separation method and application thereof, and belongs to the field of natural compound extraction and separation, wherein the extraction and separation method comprises the steps of ultrasonically extracting powder of platycodon grandiflorum by using methanol, and concentrating an extracting solution to obtain a first extract; dissolving the first extract in water, adding into adsorption resin, performing gradient elution with ethanol, and concentrating the eluate to obtain a second extract; and (3) mixing the second extract according to the volume ratio of 1: 28-32: 1: separating and purifying by 28-32 solvent system of n-hexane, ethyl acetate, methanol and water by countercurrent chromatography, and collecting eluate of polyacetylene components; concentrating the polyacetylene component effluent for the third time, and preparing by using a high performance liquid chromatograph and acetonitrile-water as a mobile phase; the method is efficient and rapid, and the purified alkyne glycoside compound has activities of resisting microorganisms, tumors and the like, and can be applied to preparation of anti-biotoxicity medicines.

The patent document with the publication number of CN106674299A relates to a polyacetylene glycoside compound and a preparation method and application thereof, wherein, dry roots of radix angelicae pubescentis are taken firstly for reflux extraction and extraction to obtain an n-butanol extraction layer; loading the n-butanol extraction layer on a silica gel column, performing gradient elution by using a chloroform-methanol system as eluent, detecting the effluent, and determining the volume ratio of the eluent to the eluent as 5: 1, and removing the solvent to obtain a first column-passing part; loading the first column chromatography part on a reverse phase silica gel chromatographic column, taking a methanol-water system as an eluent, and performing gradient elution, wherein the volume ratio of the eluent is 45: 55, and removing the solvent to obtain a second column passing part; and (3) loading the second column chromatography part on a high performance liquid chromatography separation column, and performing isocratic elution by using a mobile phase to obtain the polyacetylene glycoside compound. The invention can extract the polyacetylene glycoside compound from the radix angelicae pubescentis, and the compound can promote the uptake and transformation of glucose by cells.

The method for preparing the polyacetylene glycoside compounds cannot be well applied to the extraction of the polyacetylene glycoside compounds in the sticktight, and meanwhile, the yield of the existing sticktight polyacetylene glycoside and the sticktight polyacetylene glycoside B extracted based on the sticktight is very low, so improvement is needed.

Disclosure of Invention

The invention aims to solve the technical problem of providing a preparation process for separating and extracting the bidens bipinnata polyacetylene and the bidens bipinnata polyacetylene B aiming at the defects of the prior art so as to efficiently extract the bidens bipinnata polyacetylene and the bidens bipinnata polyacetylene B contained in the bidens bipinnata.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

the preparation process for separating and extracting the bidens bipinnata polyacetylene and the bidens bipinnata polyacetylene B comprises the following steps:

step S1: percolating herba Bidentis Bipinnatae powder by soaking in ethanol, concentrating percolate to obtain fluid extract, extracting the fluid extract with solvent, and recovering solvent under reduced pressure to obtain extract;

step S2: preparing the extract into an ethanol mixed solution, centrifuging, taking supernatant, sampling in a macroporous adsorption resin column in batches, performing gradient elution by adopting ethanol, and recovering the solvent from the first eluent after the gradient elution under reduced pressure to obtain an eluted extract;

step S3: preparing the elution extract into an ethanol mixed solution, centrifuging, taking supernatant, adding the supernatant into a polyamide column, washing with a proper amount of water, and performing gradient elution with ethanol to obtain a second eluent;

step S4: preparing the second eluent into a methanol mixed solution, loading the methanol mixed solution into a small-pore adsorption resin column in a grading manner, and performing gradient elution by adopting methanol to obtain a third eluent; separating the third eluent by Sephadex LH-20, and eluting by methanol to obtain a fourth eluent;

step S5: and concentrating the fourth eluent, taking methanol-water as a mobile phase, and preparing by adopting a high performance liquid preparation system.

Further, the percolating is carried out by soaking in ethanol, which specifically comprises the following steps: placing herba Bidentis Bipinnatae powder in percolation barrel, slowly adding 75-90% ethanol (such as 75%, 80%, 85% or 90%) until the powder is completely soaked and the ethanol level is 9-11cm lower than the medicinal material powder, sealing the percolation barrel with plastic membrane, and soaking for 24-72 hr (such as 24 hr, 36 hr, 48 hr, 64 hr or 72 hr); then the percolation is started and the ratio of (i) to (ii) is adjusted to 5-10 ml/(kg.min) [ for example: 5. collecting percolate at speed of 6, 7, 8, 9 or 10 ml/(kg.min), vacuum filtering, and supplementing 75-90% ethanol at any time to obtain total percolate. More preferably, the volume of the total percolate is 10-15 times of the weight of the bidens bipinnata in terms of L.

For the present invention, the solvents described in step S1 are petroleum ether, ethyl acetate and n-butanol.

Further, the concentration in the step S1 is to perform rotary evaporation concentration at the temperature of 40-47 ℃ until no ethanol exists, so as to obtain a fluid extract. Wherein the temperature can be 40 deg.C, 43 deg.C, 45 deg.C, 46 deg.C, 47 deg.C. Further preferably, the ratio of the volume amount of the fluid extract to the weight of the bidens bipinnata is 1L: (1-1.5) Kg.

Furthermore, the macroporous adsorption resin column is macroporous adsorption resin column D101, AB-8, HP-20 or XAD-4.

In the invention, in step S2, the extract is prepared into an ethanol mixed solution, and the extract and an ethanol solvent with the volume fraction of 15-25% are fully and uniformly mixed. Wherein, the volume fraction of the ethanol solvent can be selected from 15%, 20% and 25%.

Further, the volume concentration of ethanol in the step S2 of gradient elution with ethanol is 20-100%, for example: 20%, 40%, 60%, 80%, 100%.

For the invention, the step S3 of preparing the elution extract into an ethanol mixed solution is to fully and uniformly mix the elution extract with an ethanol solvent with the volume fraction of 8-12%. Wherein, the volume fraction of the ethanol solvent can be selected from 8%, 10% and 12%.

For the present invention, step S3 is first rinsed with an appropriate amount of water, wherein the amount of water can be routinely determined as desired, and the appropriate amount of water can preferably be 1-10 column volumes, for example: 1 column volume of water, 5 column volumes of water or 10 column volumes of water.

Further, the volume concentration of ethanol in the step S3 of gradient elution with ethanol is 5-95%, for example: 5%, 10%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 95%.

For the present invention, the step S4 of preparing the second eluent into a methanol mixed solution is to mix the second eluent and a 30-40% methanol solution by volume fraction, wherein the volume fraction of the methanol solution can be 30%, 35%, 40%.

Further, in step S4, the methanol concentration by volume in the gradient elution with methanol is 35-65% (e.g., 35%, 40%, 45%, 50%, 55%, 60%, 65%), and the methanol concentration by volume in the gradient elution with methanol is 75-85% (e.g., 75%, 80%, 85%).

Further, the volume ratio of methanol to water in the mobile phase of methanol-water is 33: 67.

bidens Bipinnata is recorded in Ben Cao Shi Yi, and it is bitter and mild in taste and non-toxic. Has the effects of clearing away heat and toxic materials, removing blood stasis, and relieving swelling. Can be used for treating swelling and pain of throat, diarrhea, malaria, dysentery, hepatitis, acute nephritis, gastralgia, dysphagia, intestinal carbuncle, traumatic injury, and snake and insect bite. The chemical components in the sticktight are complex, and the main bioactive components are flavonoids, polyacetylenes and the like.

Bidens bipinnata polyacetylene and Bidens bipinnata polyacetylene B are reported to be separated from Bidens bipinnata (Matmin. Veronica effective part chemical composition research. 2010.), but the yield of the Bidens bipinnata polyacetylene and the Bidens bipinnata polyacetylene B is extremely low and less than 0.01 ‰. Such low yield is a big barrier for the industrial application, and if the problem cannot be solved, no matter how high the medicinal value is, the wider and effective clinical application cannot be realized, and the method can only be limited to the application level of the raw material medicines. Therefore, it is critical to improve the yield of the bidens parviflora and the bidens parviflora B.

Compared with the prior art, the invention has the following beneficial effects:

1) based on the research on the characteristics of the bidens bipinnata, the invention adopts the percolation method to extract the effective components in the bidens bipinnata and obtains the percolation method extraction process and the parameters aiming at the bidens bipinnata.

2) The invention adopts three different steps of elution procedures on the bidens pilosa extract extracted by a percolation method, ensures that more impurities are removed, and improves the content of target components in the extract.

3) The yield of the target components of the bidens bipinnata polyacetylene glycoside and the bidens bipinnata polyacetylene glycoside B is high, the yield of the bidens bipinnata polyacetylene glycoside B is higher than 0.230 per mill, the yield of the bidens bipinnata polyacetylene glycoside is higher than 0.150 per mill, and the average yield is improved by more than 15 times compared with the average yield of the prior art. Wherein the yield is the weight percentage of the target component to the weight of the medicinal materials.

4) The purities of the target components of the bidens bipinnata polyacetylene B are high and can reach more than 95%.

5) The solvent used in the invention comprises ethyl acetate, ethanol and methanol, and the conventional process mostly adopts a chloroform-methanol, chloroform-methanol-water and other mixed systems, so the recovery steps are complicated, and the safety is low, therefore, the invention is safer and more environment-friendly in the use of the solvent, and has obvious effect.

6) Research shows that the alkyne glycoside compound has good biological activity of resisting microorganisms, cell toxicity, tumors and the like, and can be applied to preparing anti-biotoxicity medicines; the extraction and separation method can efficiently, environmentally and safely purify the alkyne glycoside compounds of the bidens bipinnata, namely the bidens bipinnata polyacetylene and the bidens bipinnata polyacetylene B, and can further meet the market demand on the alkyne glycoside compounds.

Drawings

FIG. 1 is a drawing showing Compound 1 of the present invention¹ An H-NMR spectrum;

FIG. 2 shows Compound 1 of the present invention¹³A C-NMR spectrum;

FIG. 3 is an HMBC profile of compound 1 of the present invention;

FIG. 4 is a drawing showing Compound 2 of the present invention¹³C-NMR spectrum.

Detailed Description

In order to better understand the present invention, the following examples are further provided to clearly illustrate the contents of the present invention, but the contents of the present invention are not limited to the following examples. In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present invention. It will be apparent, however, to one skilled in the art, that the present invention may be practiced without one or more of these specific details.

Example 1

Taking the bidens bipinnata, repeatedly crushing the bidens bipinnata by a crusher until the bidens bipinnata is completely sieved by a No. 2 sieve (pharmacopeia coarse powder), finally weighing 13.2kg, putting the bidens bipinnata in a percolation barrel, slowly adding 80% ethanol by volume until the powder is completely soaked and the liquid level of the ethanol is about 10cm higher than the medicinal material powder, stopping adding the medicinal material powder, sealing the mouth of the percolation barrel by a plastic film, and soaking for 48 hours; then starting percolation, collecting the percolate at the speed of 10 ml/(kg.min), carrying out suction filtration, and supplementing 80% ethanol at any time to obtain the total percolate, wherein the volume of the total percolate is 10 times of the weight of the bidens bipinnata in Kg in terms of L; and (3) concentrating the percolate by using a rotary evaporator at 45 ℃ until no ethanol exists to obtain a fluid extract, wherein the ratio of the volume amount of the fluid extract to the weight of the bidens bipinnata is 1L: 1Kg, extracting respectively by petroleum ether, ethyl acetate and n-butanol, replacing solvent for extraction after the color becomes lighter, recovering the solvent under reduced pressure after the extraction is finished to obtain each extraction part of the sticktight, and taking the extract of the ethyl acetate part as an extraction extract;

preparing 20% ethanol solution from the extract (203.68 g), centrifuging the solution for 5 minutes at 4000r/min in a centrifuge, taking the supernatant, and performing wet-method sample loading on a 5kg D101 macroporous adsorption resin column in a batch manner, wherein the sample loading amount is 500mL each time, after the sample loading, after all the sample solution enters the column material, performing gradient elution by using 20% -40% -60% -80% -100% ethanol respectively, and after each gradient is eluted until the effluent liquid is colorless, replacing the next gradient; merging fractions eluted by the same eluent, decompressing and recovering the solvent to obtain corresponding extract, merging the fractions after changing the gradient into the next gradient, and collecting 5 fractions in total; taking 40% ethanol fraction as eluting extract;

taking the eluted extract (87.5 g), preparing the eluted extract into a 10% ethanol solution, centrifuging the solution in a centrifuge at 4000r/min for 5 minutes, taking the supernatant, and performing wet-method sample loading on a 3kg polyamide column by times, wherein the sample loading amount is 200mL each time, after the sample loading, after the sample solution completely flows into the polyamide, flushing the polyamide with water with the volume of 1 time of the column volume, and then respectively performing elution with 10% ethanol-50% ethanol-95% ethanol, and similarly, after each gradient is eluted until the effluent liquid basically has no color, replacing the next gradient; combining fractions eluted by the same eluent, and collecting to obtain 3 fractions; taking a 10% ethanol fraction as a second eluent;

taking the second eluent, preparing into 35% methanol solution, loading about 30g of sample on a small-hole adsorption resin (MCI) column by 1kg in times with 50mL sample amount each time in the same way as the above method; then gradient elution is respectively carried out by 35 percent methanol-50 percent methanol-65 percent methanol to respectively obtain the three flow segments; taking 50% methanol fraction as third eluent; continuously separating the third eluent by sephadex SephadexLH20, eluting by 80% methanol, collecting by an automatic fraction collector, collecting 5mL of each tube, and merging according to the development result of a polyamide thin-layer chromatography plate to respectively obtain A, B, C, D, E5 fraction segments; taking the part C as a fourth eluent;

and (3) taking the fourth eluent, carrying out rotary evaporation at 45 ℃ in the dark, appropriately concentrating, and then preparing and separating active monomer components in the bidens bipinnata through a high performance liquid system, wherein the required chromatographic conditions are methanol: water = 33: 67, and then, preliminarily detecting the purity of the compound of the obtained sample at the position of 210nm of an analytical liquid phase, and after the purity reaches more than 95%, carrying out rotary evaporation at low temperature in a dark place to evaporate the solvent to dryness to obtain dry powder.

The structure of the separated compound is identified by nuclear magnetic resonance carbon spectrum and hydrogen spectrum (NMR), heteronuclear multiple carbon correlation spectrum (HMBC) of H and analysis methods and means such as consulting the research literature in the past, and 2 compounds 1 and 2 are respectively identified as Bipinata polyacetylide B and Bipinata polyacetylide B, and the structural formulas are respectively:

the compound 1 (3135.8 mg, yield 0.238 ‰) and the compound 2 (2075.6 mg, yield 0.157 ‰) are both pale yellow powders, and are assumed to be polyacetylene based on the reported chemical components.

Process for preparation of Compound 1¹The H-NMR (600 MHZ) spectrum gives the information that a trans double bond is visible in the low-field region,δ _H 6.76（1H，dt，J = 16.0，2.3 Hz），δ _H 5.91（1H，J= 16.0, 2.3 Hz), 1 glucose end group proton signalδ _H4.32（1H，d，J = 7.8 Hz）。¹³C NMR gave a signal of 19 carbons,δ _C 154.6，δ _C104.9 is the two carbon signals at the double bond;δ _C102.9 is the glucose end group carbon signal. The HMBC spectrum shows that the content of the HMBC is low,δ _H4.61 (H-1) andδ _C 80.2（C-3），δ _C70.2 (C-2) related;δ _H6.76 (H-12) andδ _C 61.4（C-13）δ _C 104.9（C-11）；δ _H4.32 (Glu-H-1) withδ _C70.3 (C-2) correlation.

Referring to FIGS. 1-3, the hydrogen and carbon spectra data are combined, and compared to literature (Matmin. Veronica needle effective site chemistry study [ D)]Shandong university of traditional Chinese medicine, 2003)), the structure of compound 1 was finally determined to be: bipinata polyacetylene glycoside B,¹³the signals of the C NMR spectrum are shown in Table 1.

Referring to fig. 4, compound 2 is similar to compound 1,¹³c NMR (150 MHZ) likewise gives a 19-carbon signal, the greatest difference from compound 1 beingδC150.12 (C-3) andδc109.37 (C-4), information on binding of Compound 1 and previous studies (study of chemical composition of effective site of Maring. spica. Meyer's patches [ D)]Shandong university of traditional Chinese medicine, 2003)), compound 2 was identified as Bipinata polyacetyleside (Bipinnata polyacetyleside), and its C-spectrum assignments are shown in Table 1:

TABLE 1 assignment of C spectra of Compounds 1, 2

Example 2

Taking the bidens bipinnata, repeatedly crushing the bidens bipinnata by a crusher until the bidens bipinnata is completely sieved by a No. 2 sieve (pharmacopeia coarse powder), finally weighing 10kg in total, placing the bidens bipinnata in a percolation barrel, slowly adding 90% ethanol by volume until the powder is completely soaked and the liquid level of the ethanol is 10cm higher than the medicinal material powder, stopping adding the medicinal material powder, sealing the percolation barrel opening by a plastic film, and soaking for 24 hours; then starting percolation, collecting the percolate at the speed of 5 ml/(kg.min), carrying out suction filtration, and supplementing 90% ethanol at any time to obtain the total percolate, wherein the volume of the total percolate is calculated by L and is 12 times of the weight of the bidens bipinnata in Kg; and (3) concentrating the percolate by using a rotary evaporator at 40 ℃ until no ethanol exists to obtain a fluid extract, wherein the ratio of the volume amount of the fluid extract to the weight of the bidens bipinnata is 1L: 1.2Kg, extracting with petroleum ether, ethyl acetate and n-butanol respectively, replacing solvent for extraction after the color becomes lighter, recovering solvent under reduced pressure after extraction is finished to obtain each extraction part of herba Bidentis Bipinnatae, and taking ethyl acetate part extract as extraction extract;

taking the extract (157.85 g), preparing into 15% ethanol solution, centrifuging the solution in a centrifuge at 4000r/min for 5 minutes, taking the supernatant, and performing wet-method sample loading on a 4kg AB-8 macroporous adsorption resin column in a fractional manner, wherein the sample loading amount is 500mL each time, after the sample loading, after all the sample solution enters the column material, performing gradient elution by respectively using 20% -40% -60% -80% -100% ethanol, and after each gradient is eluted until the effluent liquid is colorless, changing the next gradient; merging fractions eluted by the same eluent, decompressing and recovering the solvent to obtain corresponding extract, merging the fractions after changing the gradient into the next gradient, and collecting 5 fractions in total; taking 40% ethanol fraction as eluting extract;

taking the eluted extract (67.9 g), preparing the eluted extract into an 8% ethanol solution, centrifuging the solution in a centrifuge at 4000r/min for 5 minutes, taking the supernatant, and performing wet-method loading on a 2.5kg polyamide column by times, wherein the loading amount is 200mL each time, after loading, after the sample solution completely flows into the polyamide, washing the polyamide with water with the volume of 1 time of the column volume, and then respectively performing elution with 5% ethanol-50% ethanol-95% ethanol, and after each gradient is eluted until the effluent is basically colorless, replacing the next gradient; combining fractions eluted by the same eluent, and collecting to obtain 3 fractions; taking a 10% ethanol fraction as a second eluent;

taking the second eluent, preparing into 30% methanol solution, loading about 23g of sample on a small-hole adsorption resin (MCI) column by 1kg in times with 50mL sample amount each time in the same way as the above method; then gradient elution is respectively carried out by 35 percent methanol-50 percent methanol-65 percent methanol to respectively obtain the three flow segments; taking 50% methanol fraction as third eluent; continuously separating the third eluent by sephadex SephadexLH20, eluting by 75% methanol, collecting by an automatic fraction collector, collecting 5mL of each tube, and merging according to the development result of a polyamide thin-layer chromatography plate to respectively obtain A, B, C, D, E5 fraction segments; taking the part C as a fourth eluent;

and (3) taking the fourth eluent, carrying out rotary evaporation at 43 ℃ in the dark, appropriately concentrating, and then preparing and separating active monomer components in the bidens bipinnata through a high performance liquid system, wherein the required chromatographic conditions are methanol: water = 33: 67, and then, preliminarily detecting the purity of the compound of the obtained sample at the position of 210nm of an analytical liquid phase, and after the purity reaches more than 95%, carrying out rotary evaporation at low temperature in a dark place to evaporate the solvent to dryness to obtain dry powder.

The structure of the separated compound is identified by Mass Spectrometry (MS), Nuclear Magnetic Resonance (NMR) and hydrogen spectroscopy (NMR), High Performance Liquid Chromatography (HPLC) and analysis methods and means such as consulting the previous research literature, and 2 compounds 1 and 2 are respectively identified as Bipinata polyacetylide B and Bipinata polyacetylide.

The yield of the bidens pilosa polyacetylene glycoside B is 0.243 per thousand, and the yield of the bidens pilosa polyacetylene glycoside is 0.161 per thousand.

Example 3

Taking the bidens bipinnata, repeatedly crushing the bidens bipinnata by a crusher until the bidens bipinnata is completely sieved by a No. 2 sieve (pharmacopeia coarse powder), finally weighing 15kg, placing the bidens bipinnata in a percolation barrel, slowly adding 75% by volume of ethanol until the powder is completely soaked and the liquid level of the ethanol is about 10cm higher than the medicinal material powder, stopping adding the ethanol until the powder is completely soaked and the liquid level of the ethanol is close to the percolation barrel, sealing the percolation barrel opening by a plastic film, and soaking for 72 hours; then starting percolation, collecting the percolate at the speed of 8 ml/(kg.min), carrying out suction filtration, and supplementing 75% ethanol at any time to obtain the total percolate, wherein the volume of the total percolate is 15 times of the weight of the bidens bipinnata in Kg in terms of L; and (3) concentrating the percolate by using a rotary evaporator at 47 ℃ until no ethanol exists to obtain a fluid extract, wherein the ratio of the volume amount of the fluid extract to the weight of the bidens bipinnata is 1L: 1.5Kg, extracting with petroleum ether, ethyl acetate and n-butanol respectively, replacing solvent for extraction after the color becomes lighter, recovering solvent under reduced pressure after extraction is finished to obtain each extraction part of herba Bidentis Bipinnatae, and taking ethyl acetate part extract as extraction extract.

Preparing 25% ethanol solution from the extract (236.31 g), centrifuging the solution for 5 minutes at 4000r/min in a centrifuge, taking the supernatant, and performing wet-method sample loading on a 5kg HP-20 macroporous adsorption resin column in a graded manner, wherein the sample loading amount is 500mL each time, after the sample loading, after all the sample solution enters the column material, performing gradient elution by using 20% -40% -60% -80% -100% ethanol respectively, and after each gradient is eluted until the effluent liquid is colorless, changing the next gradient; merging fractions eluted by the same eluent, decompressing and recovering the solvent to obtain corresponding extract, merging the fractions after changing the gradient into the next gradient, and collecting 5 fractions in total; taking 40% ethanol fraction as eluting extract;

taking the eluted extract (101.5 g), preparing the eluted extract into a 12% ethanol solution, centrifuging the solution in a centrifuge at 4000r/min for 5 minutes, taking the supernatant, and carrying out wet-method sample loading on a 3kg polyamide column by times, wherein the sample loading amount is 200mL each time, after the sample loading, after the sample solution completely flows into the polyamide, flushing the polyamide with water with the volume of 1 time of the column volume, then respectively eluting with 10% ethanol-50% ethanol-95% ethanol, and similarly, after each gradient is eluted until the effluent liquid basically has no color, replacing the next gradient; combining fractions eluted by the same eluent, and collecting to obtain 3 fractions; taking a 10% ethanol fraction as a second eluent;

taking the second eluent, preparing into 40% methanol solution, loading about 35g of sample on a small-hole adsorption resin (MCI) column by 1kg in times with 50mL sample amount each time in the same way as the above method; then gradient elution is respectively carried out by 35 percent methanol-50 percent methanol-65 percent methanol to respectively obtain the three flow segments; taking 50% methanol fraction as third eluent; continuously separating the third eluent by sephadex SephadexLH20, eluting by 85% methanol, collecting by an automatic fraction collector, collecting 5mL of each tube, and merging according to the development result of a polyamide thin-layer chromatography plate to respectively obtain A, B, C, D, E5 fraction segments; taking the part C as a fourth eluent;

and (3) taking the fourth eluent, carrying out rotary evaporation at 47 ℃ in the dark, appropriately concentrating, and then preparing and separating active monomer components in the bidens bipinnata through a high performance liquid system, wherein the required chromatographic conditions are methanol: water = 33: 67, and then, preliminarily detecting the purity of the compound of the obtained sample at the position of 210nm of an analytical liquid phase, and after the purity reaches more than 95%, carrying out rotary evaporation at low temperature in a dark place to evaporate the solvent to dryness to obtain dry powder.

The structure of the separated compound is identified by Mass Spectrometry (MS), Nuclear Magnetic Resonance (NMR) and hydrogen spectroscopy (NMR), High Performance Liquid Chromatography (HPLC) and analysis methods and means such as consulting the previous research literature, and 2 compounds 1 and 2 are respectively identified as Bipinata polyacetylide B and Bipinata polyacetylide.

The yield of the bidens pilosa polyacetylene B is 0.245 per mill, and the yield of the bidens pilosa polyacetylene B is 0.159 per mill.

Comparative example 1

The difference from example 1 is: taking the bidens bipinnata, repeatedly crushing the bidens bipinnata by a crusher to pass through a No. 2 sieve (pharmacopeia coarse powder), taking 13.2kg in total, carrying out reflux extraction for 3 times by adopting ethanol with the volume fraction of 95%, combining extracting solutions, concentrating by a rotary evaporator at 45 ℃ until no ethanol exists, obtaining a fluid extract, wherein the ratio of the volume amount of the fluid extract to the weight amount of the bidens bipinnata is 1L: 1Kg, extracting respectively by petroleum ether, ethyl acetate and n-butanol, replacing solvent for extraction after the color becomes lighter, recovering solvent under reduced pressure after extraction is finished to obtain each extraction part of the sticktight, and taking the extract of the ethyl acetate part as the extraction extract.

The yield of the podophyllum polyacetylene glycoside B obtained by the process is 0.0314 per mill, and the yield of the podophyllum polyacetylene glycoside is 0.0127 per mill.

Comparative example 2

This comparative example was carried out by referring to the extraction and separation method described in the following document (Maming. grandma needle research on chemical composition of effective part [ D ]. Shandong university of traditional Chinese medicine, 2003.), specifically, see pages 3 to 4.

Taking 13.2kg of sticktight, crushing, extracting with 95% industrial ethanol under reflux for 3 times, mixing the extractive solutions, concentrating, removing chlorophyll, mixing with silica gel, and performing chloroform-methanol gradient elution; mixing the chloroform-methanol (10: 2) eluate with polyamide, eluting with ethanol of different concentrations, washing with water, and separating with high performance liquid phase to obtain polyglycoside B (157.73mg) and polyglycoside B (100.52 mg).

The yield of the bidens bipinnata polyacetylene glycoside B obtained by the process is 0.0119 per mill, and the yield of the bidens bipinnata polyacetylene glycoside is 0.0076 per mill.

In conclusion, the yield of the bidens bipinnata polyacetylene B and the bidens bipinnata polyacetylene prepared by the embodiment of the invention is higher, the bidens bipinnata polyacetylene B and the bidens bipinnata polyacetylene can be efficiently and environmentally extracted from the bidens bipinnata, and the two compounds are proved to have good biological activities of resisting microorganisms, cell toxicity, tumors and the like, and can be applied to the preparation of anti-biotoxicity medicines and the like.

Finally, the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting, and other modifications or equivalent substitutions made by the technical solutions of the present invention by those of ordinary skill in the art should be covered within the scope of the claims of the present invention as long as they do not depart from the spirit and scope of the technical solutions of the present invention.