Method for separating kinsenoside in anoectochilus formosanus by high-speed counter-current chromatography

Abstract

The invention discloses a method for separating kinsenoside in anoectochilus formosanus by high-speed counter-current chromatography, which comprises the following steps: firstly, extracting anoectochilus formosanus with methanol, dissolving the extract with pure water after the extract is dried by distillation under reduced pressure, extracting with chloroform, drying a water layer by distillation under reduced pressure to obtain a sample, then selecting a solvent system consisting of ethyl acetate, ethanol and water (15: 1: 15), taking the following phase as a stationary phase and the upper phase as a mobile phase, the method comprises the following steps: separating and purifying a sample by adopting a tail-to-head elution mode and using high-speed counter-current chromatography, collecting a micro fraction containing kinsenoside by using a BSZ-100 type fraction collector, finally detecting by using high performance liquid chromatography, and determining the purity of the micro fraction, according to the method, the kinsenoside in the anoectochilus formosanus can be separated in one step through high-speed counter-current chromatography, the separation purity of the kinsenoside is larger than 96%, aftertreatment is simpler, and efficient and rapid preparation of the kinsenoside is achieved.

Description

Technical Field

[0001] This invention belongs to the technical field of plant active ingredient extraction, specifically relating to a simple method for separating and purifying anoectochilin from Anoectochilus roxburghii using high-speed countercurrent chromatography. Background Technology

[0002] *Anoectochilus roxburghii*, a perennial herb belonging to the genus *Anoectochilus* in the Orchidaceae family, is also known as Golden Thread, Golden Earring, Black Ginseng, Golden Thread Tiger Head Banana, Golden Thread Bone-Eliminating Herb, and Money Grass. It is distributed in Fujian, Zhejiang, and Guangxi provinces. With its compact size, golden-yellow leaves, and reticulate veins, it is highly ornamental. *Anoectochilus roxburghii* is a traditional and precious Chinese medicinal herb with hepatoprotective, hypoglycemic, hypotensive, diuretic, and anti-tumor effects, and is safe to use due to its lack of toxic side effects. It is commonly used in traditional medicine to treat diabetes, hepatitis B, hyperlipidemia, and tumors. Its main components include glycosides, alkaloids, steroids, flavonoids, taurine, amino acids, and trace elements.

[0003] Anoectochilin is a glucopyranoside containing a chiral center and butyrolactone, which is easily hydrolyzed under acidic conditions. Wild Anoectochilus contains 16.57% anoectochilin, making it the main active ingredient. Anoectochilin has definite pharmacological activity, including hypoglycemic, hypolipidemic, hepatoprotective, and osteoporosis-improving effects. However, the supply of anoectochilin cannot meet market demand. Firstly, due to the scarcity of natural resources, traditional separation processes are often complex and inefficient, involving several steps. Secondly, the yield of artificially synthesized anoectochilin is low, and the conditions are difficult to control. Therefore, efficiently obtaining anoectochilin has undoubtedly become a challenge.

[0004] High-speed countercurrent chromatography (HSCCC), a new branch and technique in modern chromatographic separation, primarily utilizes a two-phase system within a high-speed rotating helical column to establish unidirectional fluid dynamic equilibrium between each phase. One phase serves as the stationary phase, while the other acts as the mobile phase. A constant-flow pump continuously inputs the mobile phase, and through the repeated distribution between the two phases, the column is spiraled into the sample column. Elution occurs sequentially according to the partition coefficient, retaining a large amount of stationary phase during continuous elution. On one hand, the entire separation process is repeated within a liquid-liquid distribution, avoiding direct involvement of the solid support and thus eliminating problems such as sample loss, inactivation, and denaturation caused by irreversible adsorption. This makes it particularly suitable for the separation of active ingredients from natural plants. On the other hand, as the contact between the target component and the liquid stationary phase becomes more thorough, the sample preparation volume increases. HSCCC is considered an ideal preparative separation method for the separation and purification of natural products. Compared with traditional liquid-liquid column chromatography and solid-liquid column chromatography, it offers advantages such as wide applicability, high efficiency, rapid operation, large preparation volume, low cost, and minimal sample loss. As a novel separation and purification technology, HSCCC has been widely applied in biopharmaceuticals, natural products, health foods, cosmetics, and related fields.

[0005] This invention develops a one-step high-speed countercurrent chromatography method for separating roximate from *Anoectochilus roxburghii*, which is simple, economical, environmentally friendly, and produces high-purity products. Summary of the Invention

[0006] The purpose of this invention is to provide a highly efficient method for separating and purifying Anoectochilus roxburghii glycosides.

[0007] First, *Anoectochilus roxburghii* was extracted with methanol. The extract was evaporated to dryness under reduced pressure, dissolved in pure water, and extracted with chloroform. The aqueous layer was then evaporated to dryness under reduced pressure to obtain the sample. Next, a solvent system consisting of ethyl acetate:ethanol:water (15:1:15) was selected, with the lower phase as the stationary phase and the upper phase as the mobile phase. A tail-to-head elution mode was used, and high-speed countercurrent chromatography (HSCLC) was employed to separate and purify the sample. Microfractions containing *Anoectochilus roxburghii* glycosides were collected using a BSZ-100 fraction collector. Finally, high-performance liquid chromatography (HPLC) was used for detection and purity determination.

[0008] The content of roximate obtained by the purification method of this invention is above 96%. Compared with the traditional column chromatography separation and purification method, this method is simple and convenient to operate and has high separation efficiency.

[0009] The technical solution of the present invention is as follows:

[0010] A method for separating anoectochilin from Anoectochilus roxburghii by high-speed countercurrent chromatography, comprising:

[0011] (1) Sample preparation

[0012] After the dried whole plant of Anoectochilus roxburghii was chopped, it was extracted with methanol under reflux. The extract was evaporated to dryness under reduced pressure to obtain a methanol extract. The methanol extract was dispersed in water, extracted with chloroform, and the aqueous layer was collected and concentrated to dryness under reduced pressure to obtain the sample.

[0013] The preferred material-to-liquid ratio of dried Anoectochilus roxburghii to methanol is 1:14, g / mL;

[0014] Methanol reflux extraction for 1.5 hours is preferred;

[0015] The preferred volume ratio of chloroform to water is 4:3;

[0016] (2) High-speed countercurrent chromatography separation

[0017] The sample obtained in step (1) was separated by high-speed countercurrent chromatography. The solvent system was ethyl acetate:ethanol:water volume ratio of 5-100:1:5-100. After the solvent system was mixed thoroughly, it was allowed to stand and separate into layers. The lower phase was used as the stationary phase and the upper phase was used as the mobile phase. The tail-to-head elution mode was adopted. The stationary phase was filled into the multilayer coil separation column of the high-speed countercurrent chromatograph. Then, the mobile phase was pumped into the tail end of the column at a flow rate of 0.5-5 mL / min (preferably 2 mL / min). At the same time, the high-speed countercurrent chromatograph was run at a speed of 550 rpm. When the kinetic equilibrium was reached (i.e., when there was obvious mobile phase effluent), the sample solution was injected through the sample loop. The effluent at the column head was continuously monitored with an ultraviolet detector at 190-380 nm (preferably 254 nm). The effluent containing the target compound was collected by a collector and evaporated to dryness under reduced pressure to obtain anoectochilin.

[0018] Finally, the retention of the stationary phase was determined by collecting the total column content forcibly expelled with pressurized air; the retention rate was 70%.

[0019] The sample solution is obtained by dissolving the sample obtained in step (1) in a mixture of upper and lower phases with a volume ratio of 1:1;

[0020] Two-phase solvent selection: Add 2 mg of sample to two mutually equilibrated solvent phases (1 mL each of the upper and lower phases), mix thoroughly in test tubes, and after equilibration, transfer equal volumes of the upper and lower phases to separate test tubes, remove the solvent, dilute with water (1 mL), and then analyze the concentration of roxithrin in each phase using high-performance liquid chromatography (HPLC) to determine the partition coefficient (K), K = C. L / C U C L The concentration of rosinase in the lower phase, C U The concentration of rosin in the upper phase is 1.85. When the volume ratio of ethyl acetate:ethanol:water is 15:1:15, the partition coefficient is optimal, and this is preferred as the solvent system for high-speed countercurrent chromatography analysis.

[0021] HSCCC peak components can be analyzed and identified by high-performance liquid chromatography, as detailed below:

[0022] High performance liquid chromatography (HPLC) was used to detect the eluent under the following chromatographic conditions: Venus ILMPC18 column (250 mm × 4.6 mm, 5 μm), mobile phase methanol-acetonitrile-water (1:1:9, v / v / v), flow rate 1 mL / min, wavelength 215 nm, and injection volume 20 μL.

[0023] The chromatographic peak of anoectochilin was determined by comparing its retention time with that of a standard in high performance liquid chromatography, and its purity was also determined.

[0024] The innovation and beneficial effects of this invention are as follows:

[0025] This invention provides a highly efficient method for separating and purifying Anoectochilus roxburghii glycosides. Compared with traditional separation methods, this method has the advantages of wide applicability, flexible operation, high efficiency, speed, large preparation volume, and low cost.

[0026] In the prior art, CN201010520188.4 reports a method for preparing crocin, and CN201110151736.5 reports a method for preparing crocin-I using high-speed countercurrent chromatography, both using solvent systems with different proportions of ethyl acetate, ethanol, and water. However, in the above documents, the upper phase is used as the stationary phase and the lower phase is used as the mobile phase for separation.

[0027] This invention also uses a solvent system of ethyl acetate, ethanol, and water, but unlike the aforementioned documents, it uses the lower phase as the stationary phase and the upper phase as the mobile phase to separate roximate from achyranthes bidentata glycosides. This separation method has not been previously reported. However, following the method described in the aforementioned documents, due to the inherent properties of roximate from achyranthes bidentata glycosides, the obtained roximate content is low and the purity is low, making it impossible to directly obtain pure roximate from achyranthes bidentata glycosides. (See liquid chromatogram for...) Figure 4 (B)

[0028] This invention uses high-speed countercurrent chromatography to separate roximate from Anoectochilus roximate in one step, with a roximate purity greater than 96%. Post-processing is simpler, and roximate is prepared efficiently and rapidly. Attached Figure Description

[0029] Figure 1 Chemical structure of Anoectochiloside.

[0030] Figure 2 Chromatogram of a *Anoectochilus roxburghii* glycoside sample purified by high-speed countercurrent chromatography. Solvent system: ethyl acetate:ethanol:water (15:1:15, v / v / v); stationary phase: aqueous lower phase; mobile phase: organic upper phase; flow rate: 2.0 mL / min; rotation speed: 550 rpm; sample solution: 135 mg of sample dissolved in 10 mL of a 1:1, v / v mixture of the upper and lower phases; stationary phase retention: 70%.

[0031] Figure 3 Chromatograms of samples and high-performance liquid chromatography (HPLC) analysis of microfractions separated and purified by high-speed countercurrent chromatography. (A) Chromatogram of Anoectochilin standard, (B) HPLC chromatogram of sample used for HSCCC, (C) HPLC chromatogram of Anoectochilin after separation and purification from HSCCC.

[0032] Figure 4Chromatograms of HSCCC separation and purification of micro-fractions using high-performance liquid chromatography (HPLC). (A) Chromatogram of Anoectochilin standard; (B) Chromatogram of separation and purification of micro-fractions using high-speed countercurrent chromatography with ethyl acetate, ethanol, and water (15:1:15, v / v / v), upper phase as stationary phase and lower phase as mobile phase; (C) Chromatogram of separation and purification of micro-fractions using high-speed countercurrent chromatography with ethyl acetate, ethanol, and water (15:1:15, v / v / v), lower phase as stationary phase and upper phase as mobile phase. Detailed Implementation

[0033] The present invention is further described below through specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0034] In the following examples, the whole plant of *Anoectochilus roxburghii* was obtained from Fujian Linyang Agricultural Technology Co., Ltd.

[0035] The high-performance liquid chromatography system used was a Shimadzu LC-20A analytical high-performance liquid chromatograph (Shimadzu, Japan), which consisted of an SPD-20A UV detector, dual LC-20ATvp liquid pumps, a CTO-10ASvp column oven, a LabSolutions chromatography workstation, and a 20μL injection coil.

[0036] Countercurrent chromatograph (Zhejiang University of Technology, Hangzhou). The main unit consists of two multilayer coils connected in series (PTFE tube: 1.8 mm in diameter; total volume 105 mL) and a 20 mL sample loop. High-performance liquid chromatography system (HPLC; LC-20A, Shimadzu, Japan) consists of a UV detector (SPD-20AUV), column oven (CTO-10ASvp), high-pressure binary pump (LC-20ATvp), 20 μL sample injector, and Shimadzu laboratory solution chromatography workstation. The eluent from the HSCCC column was collected using a BSZ-100 fraction collector (Shanghai Huxi Technology, Shanghai, China).

[0037] Example 1:

[0038] (1) Chop 2g of dried whole herb of Anoectochilus roxburghii, reflux with 28mL of methanol for 1.5 hours, cool to room temperature and filter, and evaporate the filtrate to dryness using a rotary evaporator to obtain methanol extract.

[0039] (2) Disperse 0.578g of methanol extract in 15ml of water, then extract with 20ml of chloroform. Concentrate the aqueous layer under reduced pressure until anhydrous to obtain the sample. (See sample HPLC chromatogram for details.) Figure 3 (B)

[0040] (3) High-speed countercurrent chromatography separation: Ethyl acetate:ethanol:water (15:1:15) was used as the solvent system. The system was thoroughly equilibrated in a separation funnel at 25°C through repeated vigorous shaking. The two phases were separated immediately before use. A tail-to-head elution mode was used, with the lower aqueous phase completely packed into a multilayer spiral column as the stationary phase. The upper organic phase was then pumped into the column tail at a flow rate of 2 mL / min, while the equipment was run at 550 rpm. After kinetic equilibrium was reached, 136 mg of the obtained sample was dissolved in 10 mL of a solution containing the solvent system in a 1:1, v / v ratio of the upper and lower phases, and injected through the sample loop. The eluent at the column head was continuously monitored at 254 nm using a UV detector. The eluent was collected for 72–96 min using a BSZ-100 fractional collector, and the solvent was evaporated under reduced pressure to obtain pure *Anoectochilus roxburghii* glycosides. Finally, the retention of the stationary phase was determined by collecting the total column content forcibly expelled by pressurized air; the retention rate was 70%. (See high-speed countercurrent chromatogram for...) Figure 2 )

[0041] (4) High-performance liquid chromatography (HPLC) analysis and identification of HSCCC peak components: The pure anoectochilin obtained in (3) was dissolved in 1 ml of pure water, and the purity of anoectochilin was detected by HPLC. The following chromatographic conditions were used: Venus ILMPC18 column (250 mm × 4.6 mm, 5 μm), mobile phase was methanol-acetonitrile-water (1:1:9, v / v / v), flow rate was 1 mL / min, wavelength was 215 nm, and injection volume was 20 μL. The anoectochilin chromatographic peak was determined by comparing the retention time with the anoectochilin standard in HPLC, and its purity was determined to be 96.7%. 11 mg of anoectochilin was obtained by lyophilization.

[0042] Example 2:

[0043] (1) The sample preparation method is the same as in Example 1.

[0044] (2) High-speed countercurrent chromatography separation: Ethyl acetate:ethanol:water (15:1:15) was used as the solvent system. The system was thoroughly equilibrated in a separation funnel at 25°C through repeated vigorous shaking. The two phases were separated immediately before use. A tail-to-head elution mode was used, with the lower aqueous phase completely packed into a multilayer spiral column as the stationary phase. The upper organic phase was then pumped into the column tail at a flow rate of 2 mL / min, while the instrument was run at 550 rpm. After kinetic equilibrium was reached, 272 mg of the obtained sample was dissolved in 10 mL of a solution containing the solvent system in a 1:1, v / v ratio of the upper and lower phases, and injected through the sample loop. The eluent at the column head was continuously monitored at 254 nm using a UV detector. The eluent was collected for 72–96 min using a BSZ-100 fractional collector, and the solvent was evaporated under reduced pressure to obtain pure *Anoectochilus roxburghii* glycosides. Finally, the retention of the stationary phase was determined by collecting the total column content forcibly expelled by pressurized air; the retention rate was 70%.

[0045] (4) High-performance liquid chromatography (HPLC) analysis and identification of HSCCC peak components: The purity of roximate was determined using HPLC. The following chromatographic conditions were used: Venus ILMPC18 column (250 mm × 4.6 mm, 5 μm), mobile phase: methanol-acetonitrile-water (1:1:9, v / v / v), flow rate: 1 mL / min, wavelength: 215 nm, injection volume: 20 μL. The chromatographic peak of roximate was determined by comparing its retention time with that of roximate standard in HPLC, and its purity was determined to be 96.2%. 19.8 mg of roximate was obtained by lyophilization.

[0046] Example 3:

[0047] (1) Chop 2g of dried Anoectochilus roxburghii whole herb sample, extract with 28mL of methanol by ultrasonic extraction for 1.5 hours, filter the extract, cool to room temperature, and evaporate the filtrate to dryness using a rotary evaporator to obtain methanol extract.

[0048] (2) Disperse 0.423g of methanol extract in 15ml of water, then extract with 20ml of chloroform. Concentrate the aqueous layer under reduced pressure until anhydrous to obtain the sample.

[0049] (3) High-speed countercurrent chromatography separation: Ethyl acetate:ethanol:water (15:1:15) was used as the solvent system. The system was thoroughly equilibrated in a separation funnel at 25°C through repeated vigorous shaking. The two phases were separated immediately before use. A tail-to-head elution mode was used, with the lower aqueous phase completely packed into a multilayer spiral column as the stationary phase. The upper organic phase was then pumped into the column tail at a flow rate of 2 mL / min, while the equipment was run at 550 rpm. After kinetic equilibrium was reached, 107 mg of the obtained sample was dissolved in 10 mL of a solution containing the solvent system in a 1:1, v / v ratio of the upper and lower phases, and injected through the sample loop. The eluent at the column head was continuously monitored at 254 nm using a UV detector. The eluent was collected for 72–96 min using a BSZ-100 fractional collector, and the solvent was evaporated under reduced pressure to obtain pure *Anoectochilus roxburghii* glycosides. Finally, the retention of the stationary phase was determined by collecting the total column content forcibly expelled by pressurized air; the retention rate was 70%.

[0050] (4) High-performance liquid chromatography (HPLC) analysis and identification of HSCCC peak components: The purity of roximate was determined using HPLC. The following chromatographic conditions were used: Venus ILMPC18 column (250 mm × 4.6 mm, 5 μm), mobile phase: methanol-acetonitrile-water (1:1:9, v / v / v), flow rate: 1 mL / min, wavelength: 215 nm, injection volume: 20 μL. The chromatographic peak of roximate was determined by comparing its retention time with that of roximate standard in HPLC, and its purity was determined to be 96.65%. 7 mg of roximate was obtained by lyophilization.

[0051] Example 4:

[0052] (1) Chop 2g of dried Anoectochilus roxburghii whole herb sample, extract with 28mL of ethanol by ultrasonic extraction for 1.5 hours, filter the extract, cool to room temperature, and evaporate the filtrate to dryness using a rotary evaporator to obtain the ethanol extract.

[0053] (2) Disperse 0.374g of ethanol extract in 15ml of water, then extract with 20ml of chloroform. Concentrate the aqueous layer under reduced pressure until anhydrous to obtain the sample.

[0054] (3) High-speed countercurrent chromatography separation: Ethyl acetate:ethanol:water (15:1:15) was used as the solvent system. The system was thoroughly equilibrated in a separation funnel at 25°C through repeated vigorous shaking. The two phases were separated immediately before use. A tail-to-head elution mode was used, with the lower aqueous phase completely packed into a multilayer spiral column as the stationary phase. The upper organic phase was then pumped into the column tail at a flow rate of 2 mL / min, while the instrument was run at 550 rpm. After kinetic equilibrium was reached, 90 mg of the obtained sample was dissolved in 10 mL of a solution containing the solvent system in a 1:1, v / v ratio of the upper and lower phases, and injected through the sample loop. The eluent at the column head was continuously monitored at 254 nm using a UV detector. The eluent was collected for 72–96 min using a BSZ-100 fractional collector, and the solvent was evaporated under reduced pressure to obtain pure *Anoectochilus roxburghii* glycosides. Finally, the retention of the stationary phase was determined by collecting the total column content forcibly expelled by pressurized air; the retention rate was 70%.

[0055] (4) High-performance liquid chromatography (HPLC) analysis and identification of HSCCC peak components: The purity of roximate was determined using HPLC. The following chromatographic conditions were used: Venus ILMPC18 column (250 mm × 4.6 mm, 5 μm), mobile phase: methanol-acetonitrile-water (1:1:9, v / v / v), flow rate: 1 mL / min, wavelength: 215 nm, injection volume: 20 μL. The chromatographic peak of roximate was determined by comparing its retention time with that of roximate standard in HPLC, and its purity was determined to be 96.7%. 5 mg of roximate was obtained by lyophilization.

[0056] Comparative example:

[0057] (1) Chop 2g of dried whole herb of Anoectochilus roxburghii, reflux with 28mL of methanol for 1.5 hours, cool to room temperature and filter, and evaporate the filtrate to dryness using a rotary evaporator to obtain methanol extract.

[0058] (2) Disperse 0.578g of methanol extract in 15ml of water, then extract with 20ml of chloroform. Concentrate the aqueous layer under reduced pressure until anhydrous to obtain the sample. (See sample HPLC chromatogram for details.) Figure 3 (B)

[0059] (3) High-speed countercurrent chromatography separation: Ethyl acetate:ethanol:water (15:1:15) was used as the solvent system. The system was thoroughly equilibrated in a separation funnel at 25°C through repeated vigorous shaking. The two phases were separated immediately before use. The upper organic phase was completely packed into a multilayer spiral column as the stationary phase. The lower aqueous phase was then pumped into the column tail at a flow rate of 2 mL / min, while the equipment was run at 550 rpm. After kinetic equilibrium was reached, 136 mg of the obtained sample was dissolved in 10 mL of a solution containing the solvent system in a 1:1 (v / v) ratio of the upper and lower phases, and injected through the sample loop. The eluent at the column head was continuously monitored at 254 nm using a UV detector. The eluent was collected using a BSZ-100 fractional collector, and the solvent was evaporated to dryness under reduced pressure.

[0060] (4) High-performance liquid chromatography (HPLC) analysis and identification of HSCCC peak components: The sample obtained in (3) was dissolved in 1 ml of pure water, and the anabolin was detected by HPLC. The following chromatographic conditions were used: Venus ILMPC18 column (250 mm × 4.6 mm, 5 μm), mobile phase was methanol-acetonitrile-water (1:1:9, v / v / v), flow rate was 1 mL / min, wavelength was 215 nm, and injection volume was 20 μL. The anabolin chromatographic peak was determined by comparing the retention time with that of the anabolin standard in HPLC.

[0061] Results: Using ethyl acetate:ethanol:water (15:1:15) as the solvent system, with the upper phase as the stationary phase and the lower phase as the mobile phase, low levels and low purity of roxithrin were obtained, making it impossible to directly obtain pure roxithrin. (See HPLC chromatogram for details.) Figure 4 (B)

Claims

1. A method for separating roximate from *Anoectochilus roxburghii* by high-speed countercurrent chromatography, characterized in that, The method includes: (1) Sample preparation After the dried whole plant of Anoectochilus roxburghii was chopped, it was extracted with methanol under reflux. The extract was evaporated to dryness under reduced pressure to obtain a methanol extract. The methanol extract was dispersed in water, extracted with chloroform, and the aqueous layer was collected and concentrated to dryness under reduced pressure to obtain the sample. (2) High-speed countercurrent chromatography separation The sample obtained in step (1) was separated by high-speed countercurrent chromatography. The solvent system was ethyl acetate:ethanol:water in a volume ratio of 5-100:1:5-100. After the solvent system was thoroughly mixed, it was allowed to stand and separate into layers. The lower phase was used as the stationary phase and the upper phase was used as the mobile phase. The tail-to-head elution mode was adopted. The stationary phase filled the multilayer coil separation column of the high-speed countercurrent chromatograph. Then, the mobile phase was pumped into the tail end of the column at a flow rate of 0.5-5 mL / min. At the same time, the high-speed countercurrent chromatograph was run at a speed of 550 rpm. When the kinetic equilibrium was reached, the sample solution was injected through the sample loop. The eluent at the column head was continuously monitored with an ultraviolet detector at 190-380 nm. The eluent containing the target compound was collected and evaporated to dryness under reduced pressure to obtain anoectochilin.

2. The method for separating roximate from *Anoectochilus roxburghii* by high-speed countercurrent chromatography as described in claim 1, characterized in that, In step (1), the ratio of dried Anoectochilus roxburghii to methanol is 1:14, g / mL.

3. The method for separating roximate from *Anoectochilus roxburghii* by high-speed countercurrent chromatography as described in claim 1, characterized in that, In step (1), methanol is refluxed for 1.5 h.

4. The method for separating roximate from *Anoectochilus roxburghii* by high-speed countercurrent chromatography as described in claim 1, characterized in that, In step (1), the volume ratio of chloroform to water is 4:

3.

5. The method for separating roximate from *Anoectochilus roxburghii* by high-speed countercurrent chromatography as described in claim 1, characterized in that, In step (2), the solvent system is ethyl acetate:ethanol:water in a volume ratio of 15:1:

15.

6. The method for separating roximate from *Anoectochilus roxburghii* by high-speed countercurrent chromatography as described in claim 1, characterized in that, In step (2), the mobile phase is pumped into the end of the column at a flow rate of 2 mL / min.

7. The method for separating roximate from *Anoectochilus roxburghii* by high-speed countercurrent chromatography as described in claim 1, characterized in that, In step (2), the sample solution is obtained by dissolving the sample obtained in step (1) in a mixture of upper and lower phases with a volume ratio of 1:

1.

8. The method for separating roximate from *Anoectochilus roxburghii* by high-speed countercurrent chromatography as described in claim 1, characterized in that, In step (2), the liquid flowing out of the column head is continuously monitored at 254 nm using an ultraviolet detector.

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