CN107033198B

CN107033198B - Method for separating chemical components of pine needles

Info

Publication number: CN107033198B
Application number: CN201610079118.7A
Authority: CN
Inventors: 柯潇; 叶亮; 张秀莉; 梁鑫淼; 赵曼茜
Original assignee: CHENGDU KANGHONG PHARMACEUTICAL CO LTD
Current assignee: CHENGDU KANGHONG PHARMACEUTICAL CO LTD
Priority date: 2016-02-04
Filing date: 2016-02-04
Publication date: 2020-06-02
Anticipated expiration: 2036-02-04
Also published as: CN107033198A

Abstract

The invention relates to a separation method of chemical components of pine needles, wherein the chemical components are betulin glucoside and massoninoside C, and the separation method is characterized by comprising the following steps: the separation method reduces the usage amount of the organic solvent, and the solvent is easy to recycle and is simple and easy to enlarge.

Description

Method for separating chemical components of pine needles

Technical Field

The invention relates to the field of medicinal chemistry, in particular to a separation method of chemical components of pine needles.

Background

Pine needles are the most major by-products of pine family (Pinaceae) plants, and represent the medicinal parts of pine. The Pinaceae plants in China mainly comprise Pinus massoniana, Chinese pine, Pinus armandii, Pinus sylvestris, slash pine and the like, wherein the Pinus massoniana is the most important pine plant in the south of China. The research in the last hundred years shows that the pine needle extract has a series of pharmacological activities of analgesia, anti-inflammation, sedation, cough relieving, phlegm eliminating, asthma relieving, blood fat reducing, oxidation resistance, aging resistance, mutation resistance, tumor resistance and the like, and the intensive research on the chemical components of the pine needles by students is caused due to rich resources and various efficacies of the pine needle extract. In recent years, the research on chemical components of the main drug in pine needles focuses on volatile oil, flavonoids, procyanidine, lignan, shikimic acid, vitamins, phenols and other substances; the separation and preparation of representative high-purity compound monomers in the pine needles becomes an important research content, and especially the extraction and separation of part of special chemical components in the pine needles has important significance for the quality research of pine needle medicinal materials. At present, the separation method of monomer chemical components in pine needles is mainly to extract and separate from plant raw materials, the related separation means is based on silica gel and gel column chromatography, and the monomer compounds prepared by the traditional methods have low purity, complex steps, long period and poor repeatability, and are difficult to apply to industrial mass production. Therefore, the separation method of the compound monomer in the pine needles needs to be improved urgently, and a separation means with rapidness, high efficiency and good reproducibility is needed to meet the requirement on the preparation of the high-purity monomer compound in the pine needles.

Disclosure of Invention

The invention aims to provide a method for extracting and separating monomer compounds from pine needles simply, quickly and efficiently, which adopts the following technical scheme:

the invention provides a separation method of chemical components of pine needles, wherein the chemical components are betulin glucoside and massonianoside C, and the specific method comprises the following steps:

(1) preparation of a test solution: weighing appropriate amount of folium Pini extract, adding organic solvent for extraction, standing the extractive solution for precipitation, collecting supernatant, concentrating, freeze drying, dissolving the dried product in ultrapure water, filtering, and collecting filtrate;

(2) separation: taking the sample solution in the step (1), performing gradient elution by taking a polar reverse-phase octadecylsilane chromatographic column as a stationary phase and a methanolic solution containing formic acid-aqueous solution containing formic acid as a mobile phase, and respectively collecting fraction F1 with the retention time of 8.0-9.0min and fraction F2 with the retention time of 20.5-22 min;

(3) and (3) purification: taking fractions F1 and F2 in the step (2), respectively taking a reversed-phase octadecylsilane chromatographic column as a stationary phase and an acetonitrile solution containing formic acid-aqueous solution containing formic acid at a ratio of 20:80 as a mobile phase, isocratically eluting, and respectively collecting fraction F1-1 with retention time of 3.8-4.6min and fraction F2-1 with retention time of 14.6-15.5 min;

(4) separation: taking fraction F1-1 and fraction F2-1 in the step (3), respectively taking a hydrophilic filler chromatographic column as a stationary phase and a 90:10 acetonitrile solution containing formic acid-aqueous solution containing formic acid as a mobile phase, carrying out isocratic elution, and collecting fraction F1-2 with retention time of 12.3-12.7min in F1-1 and fraction F2-2 with retention time of 16.7-17.3min in F2-1; wherein F1-2 is betulinoside, and F2-2 is massonianoside C.

Wherein the methanol solution containing formic acid in the step (2) is preferably a methanol solution containing 0.1% formic acid; the aqueous solution containing formic acid is preferably an aqueous solution containing 0.1% formic acid; the gradient elution conditions are preferably: 0-35min, 25-60% of methanol solution containing 0.1% of formic acid, 35-36min, 60-95% of methanol solution containing 0.1% of formic acid, 36-55min and 95-95% of methanol solution containing 0.1% of formic acid.

The acetonitrile solution containing formic acid in the step (3) or (4) is preferably an acetonitrile solution containing 0.1% formic acid; the aqueous solution containing formic acid is preferably an aqueous solution containing 0.1% formic acid.

The detection wavelength of the steps (2), (3) and (4) is preferably 280 nm;

the organic solvent in the step (1) is preferably a mixture of one or more selected from methanol, ethanol, acetone, acetonitrile, ethyl acetate and water, and is more preferably a methanol solution or a 70% ethanol solution; the filtration mode is preferably a microporous filter membrane, wherein the pore size of the microporous filter membrane is 0.22 μm or 0.45 μm; the extraction step is preferably reflux extraction or ultrasonic extraction.

Most preferably, the pine needle extract in the step (1) is weighed, methanol is added into the pine needle extract twice, the mixture is subjected to ultrasonic treatment for 1 hour each time, the mixture is kept stand and precipitated, supernatant liquid is taken, vacuum rotary evaporation is carried out at the temperature of 60 ℃, freeze drying is carried out after concentration, a dried product is dissolved in water, and the obtained product is filtered through a 0.45-micrometer filter membrane; wherein the mass volume ratio of the pine needle extract to methanol used in each ultrasonic extraction is 7: 100; or most preferably, 350g of pine needle extract is weighed, 70% ethanol is added, heating reflux is carried out for 1 hour, standing precipitation is carried out, supernate is taken, vacuum rotary evaporation is carried out at the temperature of 60 ℃, freeze drying is carried out after concentration, the dried product is dissolved in 400mL of ultrapure water and is filtered through a 0.45 mu m filter membrane, and the pine needle extract is obtained, wherein the mass volume ratio of the pine needle extract to the 70% ethanol is 7: 100. The polar reversed-phase octadecylsilane chromatographic column in the step (2) is preferably a chromatographic column with a silica gel surface simultaneously provided with a polar functional group and an octadecyl functional group, and qualified filler columns can be self-made or selected from commercially available product columns, such as chromatographic columns selected from Atlantis dC18 series, Ultimate AQ-C18 series, Xbridge C18 series and XAqua C18 series, which can meet the implementation purpose of the invention, wherein the effect is best in the qua XA C18 series, and the filler particle size of the chromatographic column is 5-60 mu m, preferably 10 mu m;

the reversed-phase octadecylsilane chromatographic column in the step (3) is preferably a chromatographic column with a silica gel surface bonded with an octadecyl functional group, and a commercially available finished column can be made or selected, such as a column selected from an Xterra MS series, an Inertsil ODS-SP series, a Zorbax SBC18 series, a SunAire C18 series and the like; most preferably the SunAire C18 series, the particle size of the chromatographic column packing is 5-60 μm, preferably 5 μm;

the hydrophilic filler chromatographic column in the step (4) is preferably a chromatographic column with polar functional groups bonded on the surface of Silica gel or a Silica gel chromatographic column, and can be made by self or selected from commercially available product columns, such as Atlantis HILIC series, Venusil HILIC series, XAmide series, ClickXIon series and the like, and is most preferably a ClickXIon series, and the particle size of the chromatographic column filler is 5-60 μm, preferably 10 μm.

The pine needle extract in the separation method of the invention is prepared by taking pine needles as a raw material and taking water or a water-containing alcohol solvent as an extraction solution; the applicant researches and discovers that water, 50% methanol or ethanol, 70% methanol or ethanol, 95% methanol or ethanol, methanol and ethanol can be used as the extraction solvent of the invention, and the effect of the methanol or the 70% ethanol is optimal; preferably, fresh pine leaves are selected and added with water for decoction twice, 8 times of solvent is added for the first time for decoction for 1.5 hours, and 6 times of solvent is added for decoction for 1.5 hours for the second time; concentrating the filtrate under reduced pressure to obtain extract with relative density of 1.2 at 70 deg.C; spray drying to obtain the final product.

Because the contents of betuloside and massonianoside C in pine needles are low, a general extraction and separation method is difficult to obtain high-purity monomer components, and a feasible chemical synthesis method for preparing the two compound monomers is not available at present. Although a small amount of monomer components can be separated by adopting the traditional systematic separation method for phytochemical research, the method has the disadvantages of complicated steps, extremely long separation period and extremely low yield, and cannot meet the requirements of industrial mass production. The applicant obtains the method for preparing the betulin glucoside and the massonianoside C through long-term research, the method has high separation efficiency, the product purity is up to more than 95 percent, the yield of the compound with the same purity is about 3 to 4 times of that of the compound prepared by the traditional method, and the preparation time period is only 9 percent of that of the compound prepared by the traditional method; and the effect of quick separation is more obvious along with the increase of the preparation amount. Meanwhile, the separation method greatly reduces the usage amount of the organic solvent, the separation environment is relatively closed, and the solvent is easy to recover, so that the method is more environment-friendly and safer; moreover, the invention is simple and convenient, is easy to enlarge, can realize the automatic separation of full instruments, greatly saves the time, labor and economic cost for preparing products, and is suitable for industrial mass production application. The method for separating the chemical components of the pine needles can be applied to extraction and separation of the betulin and the massoninoside C in pine needle raw materials such as masson pine, slash pine and the like, is an ideal way for preparing reference substances of the betulin and the massoninoside C, and makes up for the market blank of the two compound reference substances at present.

Detailed Description

Pine needle raw materials:

the folium Pini medicinal material is selected from folium Pini of Pinus massoniana Lamb of Pinaceae of Pinus of Fushun county of tribute city, Guangyuan city toward Tian district, and Nanjiang county of Bazhong city in Sichuan province of China.

Reagents and materials:

methanol: HPLC, 4L/bottle, Merck (Darmstadt, Germany);

acetonitrile: HPLC, 4L/bottle, Merck (Darmstadt, Germany);

formic acid: HPLC, 500 ml/vial, Sigma-Aldrich (st. louis, MO, usa);

deionized water was prepared from Milli-Q water purification system (Billerica, MA, USA);

pure water was obtained from Waohaha;

macroporous resin, supplier: chengdu Kelong chemical reagent factory, model: d101, (60-16 meshes) is larger than or equal to 90% in a range of 0.3-1.25 mm;

small pore resin gel (MCI), supplier: medu science popularization biology ltd, model: SBC MCI GEL reversed phase chromatographic packing F type, 75-150 μm;

dextran gel, supplier: GE Healthcare Life Sciences, model: sephadex G₂₅medium，50μm；

C18 bonded silica gel (ODS), supplier: sepax Technologies, Inc, model: GP-C18, 50 μm;

a chromatographic column: sunfire 50 x 150mm x 10 μm (Waters, usa); XAqua C1820 mm 10 μm (hua spectral acchmm column, china); click XIon 4.6 x 250mm x 10 μm (hua spectral acchmrom column, china); ClickXIon 50mm 10 μm (huaman acchmm column, china).

The instrument comprises the following steps:

ZNTPD-50 extracts the concentrator group: south pharmaceutical mechanical factory in the Yuenu area of Changsha, China

B-191 spray dryer: BUCHI, Switzerland

Purification plant: waters, usa. Including a 2525 binary high pressure prep pump, a 2777 autosampler, 2757 fraction collector, and a 2489 dual wavelength detector.

And (3) analytical chromatography: waters, usa. Including Alliance e2695 liquid phase systems and 2998 diode array detectors.

Liquid chromatography: agilent, usa. Includes a set 1260 of liquid phase separation and DAD detectors.

High resolution mass spectrometry: agilent, 6450 ultra-high resolution quadrupole-time-of-flight mass spectrometer (Q-Tof), usa.

Solid-state nmr: bruker Avance II 600MHz, Germany.

EXAMPLE A preparation of pine needle extract

Adding fresh pine leaves into a ZNTPD-50 extraction concentration unit, adding water, decocting twice, adding 8 times of water for the first time, decocting for 1.5 hours, adding 6 times of water for the second time, and decocting for 1.5 hours; concentrating the filtrate under reduced pressure to obtain extract with relative density of 1.2 at 70 deg.C; spray drying to obtain the final product.

Example two traditional phytochemical separation method for preparing pine needle compound

(1) Preparation of test solution

Taking 1200g of the pine needle extract, and sequentially extracting with ethyl acetate and water saturated n-butyl alcohol to respectively obtain an ethyl acetate extract, a water saturated n-butyl alcohol extract and a water layer residual extract, wherein the serial numbers of the extracts are Pr.1-3. The time is 5 d.

(3) Rough fraction

Taking Pr.2 (water saturated n-butanol extract), adsorbing by macroporous resin D101, eluting with 3 times column volume of water, then performing SBCMCI gel separation, eluting with 3 times column volume of 30% ethanol to obtain Pr.2A1, eluting with 3 times column volume of 40% ethanol to obtain Pr.2A2. The time is 3 d.

The Pr.2A2 (40% ethanol eluent) is taken to pass through a Sephadex G25 column, and 3 times of column volume of water is eluted to obtain 3 parts of Pr.2A2A, Pr.2A2B and Pr.2A2C. The time is 2 d.

(4) Finely dividing

①massonianoside C

And eluting Pr.2A2B through SBC MCI column and 20% ethanol with 3 times of column volume to obtain 1 part of Pr.2A2B1. It took 1.5 days.

② birch glucoside

And (3) carrying out gradient elution on Pr.2A2C by using an SBC MCI column and 20-30% ethanol, eluting and separating by using 3 times of column volume and 20% ethanol to obtain Pr.2A2C1, and eluting and separating by using 3 times of column volume and 30% ethanol to obtain Pr.2A2C2. The time is 2 d.

Separating Pr.2A2C2 by chromatography on C18 bonded silica gel (ODS), eluting with 3 times column volume of 20% ethanol to obtain Pr.2A2C3. It took 0.5 d.

(5) Purification of

①massonianoside C

Pr.2A2B1 was subjected to silica gel column chromatography, eluted sequentially with 3 column volumes of dichloromethane-ethanol (5: 1) and petroleum ether-acetone (1:3), and the petroleum ether-acetone (1:3) eluate was collected to give compound massonianoside C with a purity of 95% (HPLC, 37 mg). The time is 1 d.

② birch glucoside

Subjecting Pr.2A2C3 eluate to silica gel column chromatography, sequentially eluting with 3 times of column volume of dichloromethane-ethanol (5: 1) and petroleum ether-acetone (1:3), and collecting petroleum ether-acetone (1:3) eluate to obtain betulin glycoside with purity of 95% (HPLC, 93.67 mg). The time is 1 d.

Experiments prove that the problems of complex operation process, more experimental steps, large organic solvent consumption and the like exist when the massonianoside C and the birch glucoside are prepared from the pine needles by adopting a systematic phytochemical separation method, toxic reagents such as dichloromethane, petroleum ether, acetone and the like are involved, and the safety is low. Moreover, the method can separate 2 compounds individually, takes 19 days in total, has long preparation period and extremely low product yield, and has massonianoside C yield (relative to pine needle extract) of about three hundred thousand and betulinoside yield (relative to pine needle extract) of about eight ten thousand.

EXAMPLE three preparation of pine needle Compound

(1) Preparation of test solution

Weighing 35g of folium Pini extract, adding 500mL of methanol twice, performing ultrasonic treatment for 1 hr each time, standing for precipitation, collecting supernatant, performing vacuum rotary evaporation at 60 deg.C, concentrating, freeze drying, dissolving the dried product in 40mL of ultrapure water, and filtering with 0.45 μm filter membrane to obtain the final product. It took 9 h.

(2) One-dimensional preparative liquid phase separation

The instrument comprises the following steps: waters purification plant, column: XAqua C18(250 mm. times.20 mm. times.10 μm) was injected in an amount of 8mL per time. Performing gradient elution by using water (water phase) containing 0.1% formic acid and methanol (organic phase) containing 0.1% formic acid as mobile phases, wherein the elution conditions are 0-35min, 25-60% of organic phase, 35-36min, 60-95% of organic phase, 36-55min and 95-95% of organic phase, the flow rate is 20mL/min, and the detection wavelength is 280 nm. Separating the sample solution according to the above chromatographic conditions, and collecting fraction F1 with retention time of 8.0-9.0min and fraction F2 with retention time of 20.5-22 min. It took 7 h.

(3) Reverse phase octadecylsilane purification

The instrument comprises the following steps: waters purification plant, column: SunAire (150 mm. times.50 mm. times.5 μm), 20mL per sample. Mobile phase: 20:80 acetonitrile/water, double phase addition of 0.1% formic acid (v/v), isocratic elution at a flow rate of 80mL/min and a detection wavelength of 280 nm. Separating and purifying fractions F1 and F2 according to the chromatographic conditions, and respectively collecting fraction F1-1 with retention time of 3.8-4.6min and fraction F2-1 with retention time of 14.6-15.5 min. It took 3.5 h.

(3) Two-dimensional preparative liquid phase separation

The instrument comprises the following steps: waters purification plant, column: ClickXIon (250 mm. times.20 mm. times.10 μm) was injected in an amount of 8mL per time. Isocratic elution was carried out using a solution of water (aqueous phase) containing 0.1% formic acid and acetonitrile (organic phase) containing 0.1% formic acid as mobile phases, the organic phase: aqueous phase (90:10), flow rate 80ml/min, detection wavelength 280 nm. Separating fractions F1-1 and F2-1 respectively according to the above chromatographic conditions, collecting fractions with retention time of 12.5-13.5min from F1-1 to obtain birch glucoside (F1-2)11.15mg, purity 98% (HPLC), yield (relative to pine needle extract) 0.032%, and time consumption 13 h.

Fractions of F2-1 with a retention time of 16.5-17.5min were collected to give massonianoside C (F2-2)3.8mg, purity 95% (HPLC), yield (relative to pine needle extract) 0.011%, taking 17 h.

Example preparation of a four pine needle Compound

(1) Preparation of test solution

Weighing folium Pini extract 350g, adding 5L 70% ethanol, heating under reflux for 1 hr, standing for precipitation, collecting supernatant, vacuum rotary evaporating at 60 deg.C, concentrating, freeze drying, dissolving dried product in 400mL ultrapure water, and filtering with 0.45 μm filter membrane. It took 10 h.

(2) One-dimensional preparative liquid phase separation

Preparing a liquid phase instrument: waters purification plant, column: XAqua C18(250 mm. times.100 mm. times.10 μm) was injected in an amount of 40mL per sample. Gradient elution was carried out with water (aqueous phase) containing 0.1% formic acid and methanol (organic phase) containing 0.1% formic acid as mobile phases, under the following conditions: 0-35min, 25-60% of organic phase, 35-36min, 60-95% of organic phase, 36-55min and 95-95% of organic phase, wherein the flow rate is 300mL/min, the detection wavelength is 280nm, the sample solution is separated according to the chromatographic conditions, and fraction F1 with the retention time of 8.0-9.0min and fraction F2 with the retention time of 20.5-22min are respectively collected. It took 12 h.

(3) Reverse phase octadecylsilane purification

The instrument comprises the following steps: waters purification plant, column: SunAire (150 mm. times.50 mm. times.5 μm), 20mL per sample. Mobile phase: 20:80 acetonitrile/water, double phase addition of 0.1% formic acid (v/v), isocratic elution at a flow rate of 80mL/min and a detection wavelength of 280 nm. Separating and purifying fractions F1 and F2 according to the chromatographic conditions, and collecting fractions F1-1 and F2-1 with retention time of 3.8-4.6min and F2 and 14.6-15.5min in F1. It took 12 h.

(4) Two-dimensional preparative liquid phase separation

The instrument comprises the following steps: waters purification plant, column: ClickXIon (250 mm. times.50 mm. times.10 μm) was injected in an amount of 20mL per time. Isocratic elution was carried out using a solution of 0.1% formic acid in water (aqueous phase) and 0.1% formic acid in acetonitrile (organic phase) as mobile phases, the aqueous phase being 10:90, the flow rate being 80ml/min, and the detection wavelength being 280 nm. Separating fractions F1-1 and F2-1 respectively according to the above chromatographic conditions, collecting fractions with retention time of 12.5-13.5min from F1-1 to obtain betulin (F1-2)100.8mg, purity of compound 97% (HPLC), and yield (relative to folium Pini extract) 0.03%. The fraction remaining in F2-1 for 16.5-17.5min was collected to obtain massonianoside C (F2-2)35.4mg, purity 96% (HPLC), yield (relative to pine needle extract) 0.01%. It took 12 h.

EXAMPLE five Compound Structure verification

(1) Liquid phase and mass spectrometry

The compounds separated in the third and fourth examples were analyzed by Agilent 1260 liquid chromatography. A chromatographic column: ClickXIon column (4.6X 250mm,10 μm) was subjected to isocratic elution using an aqueous solution (aqueous phase) containing 0.1% formic acid and an acetonitrile solution (organic phase) containing 0.1% formic acid as mobile phases, the organic phase: aqueous phase (90:10) at a flow rate of 1.0 ml/min. The liquid phase analyte was directly coupled to Agilent 6540Q-TOF MS. The gas temperature was set at 325 ℃, the atomization gas pressure was set at 30psi, the drying gas flow rate was set at 8L/min and the capillary voltage was set at 3.5 kV. .

Purity and liquid chromatography data analysis software were Waters Empower software (version3.0) and Masslynxsoftware (version 4.1) (Milford, MA, USA)

Mass spectrometry data analysis software is Agilent Master software (version 4.0) (Santa Clara, Calif., USA)

(2) Nuclear magnetic resonance

The analysis was carried out using a Bruker Avance II 600MHz NMR spectrometer. Hydrogen spectrum: 600MHz, the sample solvent is MeOD. Carbon spectrum: 150MHz, the sample solvent is MeOD.

The 1H NMR and 13C NMR nuclear magnetic data analysis software was Bruker Topspin software (version3.0).

(3) Identification results

The spectrum, mass spectrum and nuclear magnetic data of the F1-2 (betuloside) compound are shown in the following table 1, and the molecular formula of the compound is shown in the formula I:

TABLE 1 structural identification data for Compound F1-2

The spectrum, mass spectrum and nuclear magnetic data of the F2-2(massonianoside C) compound are shown in the following table 2, and the molecular formula of the compound is shown in the formula II:

TABLE 2 structural identification data for Compound F2-2

Claims

1. A method for separating chemical components from pine needles, wherein the chemical components are betulinoside and massonianoside C, and the pine needles are pine needles of Pinus massoniana, and the method is characterized by comprising the following steps of: (1) preparation of a test solution: weighing appropriate amount of folium Pini extract, adding organic solvent for extraction, standing the extractive solution for precipitation, collecting supernatant, concentrating, freeze drying, dissolving the dried product in ultrapure water, filtering, and collecting filtrate;

(3) and (3) purification: taking fractions F1 and F2 in the step (2), respectively taking a reversed-phase octadecylsilane chromatographic column as a stationary phase and an acetonitrile solution containing formic acid-aqueous solution containing formic acid at a ratio of 20:80 as a mobile phase, isocratically eluting, and respectively collecting fraction F1-1 with retention time of 3.8-4.6min and fraction F2-1 with retention time of 14.6-15.5 min; (4) separation: taking fraction F1-1 and fraction F2-1 in the step (3), respectively taking a hydrophilic filler chromatographic column as a stationary phase and a 90:10 acetonitrile solution containing formic acid-aqueous solution containing formic acid as a mobile phase, carrying out isocratic elution, and collecting fraction F1-2 with retention time of 12.3-12.7min in F1-1 and fraction F2-2 with retention time of 16.7-17.3min in F2-1, wherein F1-2 is betulinoside, and F2-2 is massonianoside C.

2. The separation method according to claim 1, wherein the methanol solution containing formic acid in the step (2) is a methanol solution containing 0.1% formic acid.

3. The separation method according to claim 1, wherein the aqueous solution containing formic acid in the step (2) is an aqueous solution containing 0.1% formic acid.

4. The separation method according to claim 1, wherein the gradient elution conditions in step (2) are: 0-35min, 25-60% of methanol solution containing 0.1% of formic acid, 35-36min, 60-95% of methanol solution containing 0.1% of formic acid, 36-55min and 95-95% of methanol solution containing 0.1% of formic acid.

5. The separation process according to claim 1, wherein the acetonitrile solution containing formic acid in step (3) or (4) is an acetonitrile solution containing 0.1% formic acid.

6. The separation process according to claim 1, characterized in that the aqueous solution containing formic acid in step (3) or (4) is an aqueous solution containing 0.1% formic acid.

7. The separation method according to any one of claims 1 to 6, wherein the detection wavelength in the step (2) or (3) or (4) is 280 nm.

8. The separation method according to claim 1, wherein the organic solvent in step (1) is selected from one or more of methanol, ethanol, acetone, acetonitrile, and ethyl acetate.

9. The separation method according to claim 8, wherein the organic solvent in step (1) is methanol or a 70% ethanol solution.

10. The separation method according to claim 1, wherein the filtration means in step (1) is a microporous membrane.

11. The separation method according to claim 10, wherein the pore size of the microfiltration in step (1) is 0.22 μm or 0.45 μm.

12. The separation method according to claim 1, wherein the extraction step in step (1) is reflux extraction or ultrasonic extraction.

13. The separation method according to any one of claims 8 to 12, wherein the step (1) is a step of weighing the pine needle extract, adding methanol into the pine needle extract twice, performing ultrasonic treatment for 1 hour each time, standing and precipitating, taking the supernatant, performing vacuum rotary evaporation at 60 ℃, performing freeze drying after concentration, dissolving the dried product in ultrapure water, and filtering the ultrapure water through a 0.45 μm filter membrane to obtain the pine needle extract; wherein the mass volume ratio of the pine needle extract to the methanol used in each ultrasonic extraction is 7: 100.

14. The separation method according to any one of claims 8 to 12, wherein the step (1) comprises weighing 350g of the pine needle extract, adding 70% ethanol, heating and refluxing for 1 hour, standing and precipitating, taking the supernatant, performing vacuum rotary evaporation at 60 ℃, performing freeze drying after concentration, dissolving the dried product in 400mL of ultrapure water, and filtering through a 0.45 μm filter membrane, wherein the mass-volume ratio of the pine needle extract to the 70% ethanol is 7: 100.

15. The separation method according to claim 1, wherein the polar reversed-phase octadecylsilane chromatographic column in the step (2) is a chromatographic column having both a polar functional group and an octadecyl functional group on the surface of silica gel.

16. The method of claim 15, wherein the column in step (2) is an Atlantisis dC18 series, an Ultimate AQ-C18 series, an Xbridge C18 series, or an XAqua C18 series column.

17. The separation process according to claim 16, characterized in that the chromatographic column in step (2) is of the XAqua C18 series.

18. The separation method according to claim 1, wherein the size of the packing of the chromatography column in the step (2) is 5 to 60 μm.

19. The separation method according to claim 18, wherein the chromatographic column packing in step (2) has a particle size of 10 μm.

20. The separation method according to claim 1, wherein the reversed-phase octadecylsilane chromatographic column in the step (3) is a silica gel surface-bonded octadecylsilane functional group chromatographic column.

21. The separation method according to claim 20, wherein the chromatographic column in step (3) is

Xterra MS series, Inertsil ODS-SP series, Zorbax SB C18 series, or SunAire C18 series.

22. The separation method according to claim 21, wherein the chromatographic column in step (3) is

SunAire C18 series.

23. The separation method according to claim 1, wherein the size of the packing of the chromatography column in the step (3) is 5 to 60 μm.

24. The separation method according to claim 23, wherein the chromatographic column packing in step (3) has a particle size of 5 μm.

25. The separation method according to claim 1, wherein the hydrophilic filler chromatographic column in the step (4) is a chromatographic column with polar functional groups bonded on the surface of silica gel or a chromatographic column with silica gel.

26. The method of claim 25, wherein in step (4) the column is an Atlantis HILIC Silica series, Venusil HILIC series, XAmide series or ClickXIon series.

27. The separation method according to claim 26, wherein the column in step (4) is a ClickXIon series column.

28. The separation method according to claim 1, wherein the size of the packing of the chromatography column in the step (4) is 5 to 60 μm.

29. The separation method according to claim 28, wherein the chromatographic column packing in step (4) has a particle size of 10 μm.

30. The separation method according to claim 1, wherein the pine needle extract is extract powder obtained by extracting pine needles as a raw material with water or a water-containing alcohol solvent as an extraction solvent.

31. The separation method according to claim 30, wherein the pine needle extract is prepared by decocting fresh pine needles with water twice, wherein 8 times of solvent is added for the first time, and 1.5 hours is added for the second time, and 6 times of solvent is added for the second time, and the decoction time is 1.5 hours; concentrating the filtrate under reduced pressure to obtain extract with relative density of 1.2 at 70 deg.C; spray drying to obtain the final product.