CN117362307A - Preparation method and application of bergenia plant active ingredient - Google Patents

Abstract

The invention relates to the technical field of plant extraction, in particular to a preparation method and application of bergenia plant active ingredients, wherein the preparation method comprises the following steps: crude extract preparation, selection of biphasic solvent system, solvent system and sample solution preparationAnd high-speed countercurrent chromatographic separation. The present invention allows for the isolation of compounds from the rhizome of schefflera arboricola by establishing an efficient one-step internal recycle CCC process. The invention adopts an optimized biphasic solvent system to successfully separate five compounds of quercetin-rhamnoside, quercetin-3-O-rhamnoside, bergenin, kaempferol and palmitic acid, and the purity is equal>98%, the established one-step inner loop CCC method is applicable to the method with similar K _D The separation of the compound has the advantages of less solvent consumption, short time consumption, high theoretical polar plate number and the like.

Description

Preparation method and application of active ingredients of cabbage plants

Technical field

The present invention relates to the technical field of plant extraction, and specifically relates to a preparation method and application of active ingredients of cabbage plants.

Background technique

Rock seven, a plant of the genus Lithops, is a famous medicinal plant used to treat different diseases such as diabetes, diarrhea, vomiting, cough, fever, wound healing, lung diseases and cancer. Among them, 3-O-Galloylepicatechin and 3-O-galloylcatechin are two compounds isolated from the methanol extract of Pangasius. They have inhibitory effects on the activity of rat intestinal α-glucosidase and pig pancreatic α-amylase. Strong dose dependence. The medicinal effect of Pangasius is related to its large number of active ingredients, and its active ingredients can be separated using silica gel LH-20 and high-performance liquid chromatography. However, its structural similarity and high polarity make it still a big challenge to separate various components from the rhizomes of Pangasius.

Contents of the invention

The purpose of the present invention is to overcome the shortcomings of the existing technology and propose a method for preparing the active ingredients of rock cabbage plants, using an efficient one-step internal circulation countercurrent chromatography method to prepare, separate and purify compounds with similar distribution coefficients in rock seven. .

The technical solutions adopted by the present invention to solve the technical problems are:

A method for preparing active ingredients of cabbage plants, including:

Step 1. Preparation of crude extract: Take the rhizome of Pangasius, extract it with methanol, combine the extracts and concentrate to obtain the methanol crude extract of Pangasius;

Step 2. Selection of biphasic solvent system: Put the crude methanol extract sample into a container, add the balanced biphasic solvent, and shake repeatedly to make the sample completely distributed in the two phases. Let it stand and divide into two layers. Add methanol to dilute; calculate the K _D value by dividing the peak area of the target compound in the lower phase - stationary phase A _L by the peak area of the upper phase - mobile phase A _U : K _D = A _L / _AU ;

Step 3. Solvent system and sample solution preparation

Preparation for HSCCC separation: Add a biphasic solvent system containing TBME-n-butanol-acetonitrile-water to the separatory funnel and shake it vigorously, then let it stand until it reaches a two-phase equilibrium state. The upper and lower phases serve as stationary phase and mobile phase respectively. ;

Sample solution preparation process: Add the Pangasius extract into the container, and add the upper phase and lower phase of the TMBE-n-butanol-acetonitrile-water solvent system respectively to completely dissolve the sample; as an option, the added Pangasius extract should be in the same container as The volume ratio is 1: (10-90).

Step 4. High-speed countercurrent chromatography separation

One-dimensional separation: Use a six-way valve, place the six-way valve one between the solvent bottle and the pump to facilitate switching between internal circulation and collection, and connect the six-way valve two to the six-way valve one and the detector. between. This invention only uses six-way valve one to control the internal circulation;

Internal loop separation: Close the six-way valve collection mode to enter the internal loop countercurrent chromatography CCC separation process; close the six-way valve and pump the eluent back to the column to form a cycle, with another peak after the first peak of a cycle If the terminal peaks overlap, switch the cycle mode to the normal elution method, and use the elution method from beginning to end to avoid overlapping of peaks to obtain the compound.

Further, in step 1, the rhizome of Pangasius is extracted three times with methanol, the extracts are combined, and concentrated under reduced pressure and 45°C to obtain a crude methanol extract of Pangasius.

Further, in step 3, the volume ratio of TBME-n-butanol-acetonitrile-water is (1-5): (1-5): (0.5-3): (2-10), for example: The volume ratio is 2:2:1:5 or 3:1:1:5 or 1:3:1:5.

Further, in step 4, one-dimensional separation: Pump the upper phase (stationary phase) into the CCC in 30mL/min head-to-tail elution mode, adjust the CCC instrument speed to 800 rpm at 25°C, and use 2.0mL The lower phase (mobile phase) is pumped into the lower phase (mobile phase) at a speed of /min until equilibrium is achieved to obtain the appropriate stationary phase retention value (S _f value). Inject the sample after equilibration, and use a portable recorder to monitor the effluent at a wavelength of 254 nm; close the internal circulation valve before elution.

Further, the purified product includes quercetin-rhamnoside, quercetin-3-O-rhamnoside, fuscine, kaempferol and palmitic acid.

Further, after the sample is injected, switch to the circulation mode through the six-way valve. When the specific target peak reaches separation, it switches to the normal mode and collects it. After the collection is completed, it switches to the circulation mode to perform internal circulation separation of other components. Repeat the above Steps to complete the separation of compounds; the compounds kaempferol and palmitic acid are eluted in the first cycle, the compound cobracine is eluted in the second cycle, and the compounds quercetin-rhamnoside, quercetin -3-O-rhamnoside eluted in the fourth cycle.

Further, the preparation method adopts an internal circulation CCC system, which includes a six-way valve one, a six-way valve two, a pump, a solvent bottle, a sampler, a countercurrent separation column and a detector; the six-way Valve one is connected between the solvent bottle and the pump to facilitate switching between internal circulation and collection. The six-way valve two is connected between the six-way valve one and the detector; the pump is connected to the injector, and The sampler is connected to the counterflow separation column, the counterflow separation column is connected to the detector, the detector is connected to six-way valve two, six-way valve two is connected to the collection ring, six-way valve one is connected to the distillate pipe; close six-way valve one Pump the eluate back to the column to form a cycle

Application of the compounds extracted by the method for preparing active ingredients of Brassica genus plants in the medical field.

Technical effects of the present invention:

Compared with the existing technology, the present invention establishes an effective one-step internal circulation CCC method to isolate compounds from the rhizomes of Pangasius in Pakistan. Quercetin-rhamnoside and quercetin-3-O- were successfully separated using an optimized biphasic solvent system (TBME/n-butanol/acetonitrile/water (2:2:1:5, v/v) Five compounds: rhamnoside, cochinacein, kaempferol and palmitic acid. The established one-step internal cycle CCC method is suitable for the separation of compounds with similar K _D values, and has the advantages of low solvent consumption, short time consumption, high number of theoretical plates High advantages.

Description of the drawings

Figure 1 is a chemical structure diagram of the isolated compounds in the crude petrous methanol extract of the present invention;

Figure 2 is a schematic diagram of the HSCCC separation mode of the present invention, wherein Figure 2(A) is the normal elution mode, and Figure 2(B) is the internal circulation mode;

Figure 3 is a chromatogram of compound separation in the HSCCC internal circulation mode of the present invention;

Figure 4 is an HPLC chromatogram of the methanol extract of Pangasius of the present invention.

In the figure, 11. Six-way valve one; 12. Six-way valve two; 13. Solvent bottle; 14. Pump; 15. Detector; 16. Injector; 17. Countercurrent separation column; 18. Distillate pipe; 19. Collection circle.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described below in conjunction with the accompanying drawings.

Example 1:

This embodiment relates to a method for preparing active ingredients of the rock cabbage plant, extracting the compound from the rhizomes of rock seven, wherein the rock seven described in this embodiment is produced in Pakistan.

1.1 Reagents and materials

Analytical grade methanol, tert-butyl methyl ether (TBME), n-butanol and acetonitrile were used for extraction and HSCCC separation, HPLC grade acetonitrile was used for HPLC, and milli-Q (18MΩ) system pure water was used.

1.2 Equipment

Separation was performed using a model TBE-300C HSCCC device using three 300 ml multilayer coil separation columns and a 20 ml manual sample loop. The HSCCC equipment is equipped with a 254nm 8823A-UV monitor, a TBP-5002 constant flow pump, a Model 3057 portable recorder, and a cryogenic thermostatic bath to maintain a constant temperature (25°C). The HPLC equipment was a Waters 600 system, including a PDA detector 15, a Waters 600 pump and an autosampler 16, symmetrical C18 column (250 mm × 4.6 mm, i.d. 5 μm, USA). An Agilent 1100SL series MSD Trap was used for electrospray tandem mass spectrometry (ESI/MS). An electrospray ion source MSD trap was also used, and a Bruker Avance DRX-400 was used to record nuclear magnetic resonance spectra. All chemical changes are expressed in PPM as delta values compared to the endogenous control tetramethylsilane.

1.3 The preparation method includes:

Step 1. Preparation of crude extract: Rinse the collected plant rhizomes with tape and distilled water, and then dry them at room temperature in a cool place; extract the dried rhizomes (2.0kg) three times with 90% methanol, combine the extracts, and incubate under reduced pressure at 45°C. Conditions were concentrated to obtain 102g of crude methanol extract of Penthospermum;

Step 2. Selection of biphasic solvent system: After weighing, take an appropriate amount of sample and put it into a 10ml centrifuge tube. Take 1ml of each balanced biphasic solvent and put it into the centrifuge tube and shake it repeatedly to make the sample completely distributed in the two phases. Set into two layers, add 1 mL of methanol to each phase to dilute, and analyze with HPLC; calculate K by dividing the peak area of the target compound in the stationary phase (lower phase, A _L ) by the peak area of the mobile phase (upper phase, A _U ) _D value (K _D = _AL /A _U );

Step 3. Solvent system and sample solution preparation

Preparation for HSCCC separation: Add a biphasic solvent system containing TBME-n-butanol-acetonitrile-water (2:2:1:5, v/v) into the separatory funnel and shake it vigorously, and let it stand for 15 minutes. into a two-phase equilibrium state, with the upper and lower phases serving as stationary phase and mobile phase respectively;

Sample solution preparation process: Add 280 mg of Pangasius extract into a 10 ml centrifuge tube, and add 5 ml of the upper phase and 5 ml of the lower phase of the TMBE-n-butanol-acetonitrile-water (2:2:1:5, v/v) solvent system. phase to completely dissolve the sample;

Step 4. High-speed countercurrent chromatography separation

One-dimensional separation

Use a six-way valve capable of internal circulation, place the six-way valve 11 between the solvent bottle 13 and the pump 14 to facilitate switching between internal circulation and collection, and place the six-way valve 212 (since there is no need to Online storage in the external column, therefore closed) is connected between the six-way valve 11 and the detector 15 (Fig. 2A); the pump 14 is connected to the injector 16, the injector 16 is connected to the countercurrent separation column 17, and the countercurrent separation column 17 is connected to the detector 15, the detector 15 is connected to the six-way valve 12, the six-way valve 12 is connected to the collection ring 19, and the six-way valve 11 is connected to the distillate pipe 18. The present invention only uses six-way valve 11 to control the internal circulation.

Pump the upper phase (stationary phase) into the CCC in head-to-tail elution mode at 30 mL/min, adjust the CCC instrument speed to 800 rpm at 25°C, and pump the lower phase (mobile phase) at a speed of 2.0 mL/min. ) until equilibrium is obtained to obtain the appropriate stationary phase retention value (S _f value). Inject the sample after equilibrium, and use a portable recorder to monitor the effluent at a wavelength of 254 nm; close the internal circulation valve before elution; after the sample is injected, switch to the circulation mode through the six-way valve - 11, and switch to the circulation mode when the specific target peak reaches separation. In the normal mode, collect. After the collection is completed, switch to the circulation mode to perform internal circulation separation of other components. Repeat the above steps to complete the compound separation;

Inner loop separation

Close the six-way valve collection mode to enter the internal circulation countercurrent chromatography CCC separation process. Close the six-way valve 11 and pump the eluate back to the column to form a cycle (as shown in Figure 2B). After the first peak of a cycle overlaps with the end peak of another peak, convert the cycle mode to the normal elution method. Use a sequential elution method from beginning to end to avoid overlapping peaks to obtain purified products;

Step 5. HPLC analysis and structural identification

A Water 600 system was used for chromatographic separation. At a column temperature of 25°C and a flow rate of 1.0 mL/min, the column was eluted with a mobile phase consisting of water (A) and acetonitrile (B) with the following gradient: 0-7 min, 90-70%A; 7-12min, 70-65%A; 12-13min, 65-90%A, 13-20min, 90%A. Obtain the spectrum at 190~400nm and the chromatogram at 254nm.

ESI-MS analysis was performed using an Agilent 6520 Q-TOF instrument (Agilent, Santa Clara, CA, USA), CD ₃ OD was used as the solvent, and NMR spectra were recorded using Bruker Biospin (Rheinstetten, Germany), expressed in parts per million (ppm). ) plus a constant (J) represents the chemical shift (δ) in Hz.

1.4 Results and discussion

1.4.1 Selection of biphasic solvent system

It is very important to choose an appropriate biphasic solvent system when using HSCCC separation. The appropriate solvent should have an appropriate distribution coefficient and good sample solubility. This example examined several mixed solvent systems, including ethyl acetate/n-butanol/water (4:1:5, v/v), n-hexane/ethyl acetate/methanol/water (0.5:4.5:0.5 :4.5, v/v), chloroform/methanol/water (5:2:3, v/v) and TBME/n-butanol/acetonitrile/water (2:2:1:5, v/v) (Table 1 ). If ethyl acetate/n-butanol/water (4:1:5, v/v) and n-hexane/ethyl acetate/methanol/water (0.5:4.5:0.5:4.5, v/v) are used, these compounds Mainly distributed in the upper phase, the K _D value is large, which increases the difficulty of elution. If chloroform/methanol/water (5:2:3, v/v) is used, the compounds are mainly distributed in the lower phase, which results in fast elution and low peak resolution.

The TBME/n-butanol/acetonitrile/water solvent system is used to separate compounds of moderate to high polarity. In the solvent system, increasing the ratio of acetonitrile to TBME will lead to the formation of three phases, so the potential ratio is limited. When using TBME/n-butanol/acetonitrile/water (1:3:1:5, v/v) and (2:3:0:5, v/v) solvent systems, peak elution starts very late, with The peaks were not eluted well. The smaller K _D values of compounds 1 and 2 when using the TBME/n-butanol/acetonitrile/water (3:1:1:5, v/v) solvent system indicate poor peak resolution. Compared with the tested solvent system, TBME/n-butanol/acetonitrile/water (2:2:1:5, v/v) is preferably in the range of 0.87 to 1.79 (Table 1). The distribution coefficients of compounds 1 and 2 are The partition coefficients of compounds 3, 4, and 5 are less than 1.50. The low distribution coefficient indicates that it is difficult to separate these compounds by traditional one-step separation technology, so this example adopts the internal circulation mode in HSCCC.

Table 1 Distribution coefficients of compounds in countercurrent chromatography solvent systems

1.4.2 Separation of chemical components

Using TBME/n-butanol/acetonitrile/water solvent system (2:2:1:5, v/v) in conventional HSCCC with a sample load of 280 mg, compounds 3, 4 and 5 could be separated in the first step of separation. However, the separation rate is poor (Figure 3). In this experiment, the compound was recycled and a better separation rate was obtained. In the first cycle, compounds 4 (37 mg) and 5 (43 mg) were eluted with appropriate K _D values. After HPLC analysis, their purity was greater than 98%. The uniqueness of this method is that in the first cycle Two pure compounds are separated in this cycle. If compounds 4 and 5 are not eluted in the first cycle, the compounds will enter the second cycle, and then the compounds can merge with each other, which increases the complexity of the separation. Therefore, compounds 4 and 5 eluted in the first cycle, compound 3 (76 mg) eluted in the second cycle, and compounds 1 (33 mg) and 2 (36 mg) eluted in the fourth cycle. The most suitable separation solution. According to HPLC analysis, the purity of these compounds was greater than 98% (Figure 4).

1.4.3 Internal circulation countercurrent chromatography program

The basic equation of chromatographic separation is the relationship between resolution, number of theoretical plates and column length during separation.

(R ₁ /R ₂ ) ² ＝N ₁ /N ₂ ＝L ₁ /L ₂

R represents the resolution, N represents the total theoretical number of plates, and L represents the column length. The longer the CCC column, the greater the number of theoretical plates and better separation rate. HSCCC has fewer theoretical plates compared to HPLC, so the value of CCC separations is lower, however its value can be increased by fine-tuning the inner loop in the classic HSCCC method, which results in similar separation K _D values in phytochemical separations relatively complex compounds are possible.

In the internal cycle CCC mode, the last peak of one cycle overlaps with the first peak of the second cycle. Compounds begin to overlap in the second cycle and pure compounds are eluted. Sequential elution of two compounds in the first cycle and one compound in the second and third cycles facilitates the isolation of pure compounds. Compared with the traditional long-time elution process, the circulation mode has advantages in terms of solvent usage and time consumption. It increases the number of theoretical plates N, improves peak resolution, is simple to set up, and is easy to operate.

1.4.4 Structural identification

Quercetin-rhamnoside (1): ESI-MS (positive ion mode) m/z 773.0314[M+H] ⁺ , ¹ H-NMR (400MHz, DMSO-d ₆ ): δ _H 6.21 (1H,d ,arom,H-6),6.43(1H,d,arom,H-8),7.70(1H,d,arom,H-2'),6.87(IH,d,H-5'),7.63(1H ,dd,arom,H-6'),5.09(1H,d,H-1”),3.47(1H,m,H-2”),3.39(1H,m,3”),3.38(1H,m ,4”),3.35(1H,m,5”),3.77(1H,dd,H-6”A),3.79(1H,m,H-6”B),4.55(1H,dd,1”'),3.93(1H,dd,H-2”'),3.60(1H,dd,H-3”’),3.46(1H,m,H-4”’),3.52(1H,dd,H-5”'),1.11(1H,d,H-6”’),4.41(1H,d,H-1””),3.27(1H,m,H-2””),3.40(1H,m,H -3””),3.40(1H,m,H-4””),3.23(1H,m,H-5””),3.71(1H,dd,H-6””). ¹³ C-NMR( 100MHz DMSO-d ₆ ): δ _C 160.3(C-2), 135.3(C-3), 180.0(C-4), 162.3(C-5), 100.3(C-6), 166.0(C-7) ,95.1(C-8),159.0(C-9),104.7(C-10),122.6(C-1'),118.2(C-2'),145.6(C-3'),150.0(C- 4'),115.6(C-5'),123.6(C-6'),105.3(C-1”),76.0(C-2”),77.0(C-3”),72.0(C-4” ),78.5(C-5”),69.3(C-6”),101.8(C-1”’),70.8(C-2”’),83.0(C-3”’),72.0(C-4 ”'),69.5(C-5”’),17.8(C-6”’),105.6(C-1””),75.6(C-2””),77.6(C-3””),71.0 (C-4””),77.6(C-5””),62.0(C-6””).

Quercetin-3-O-rhamnoside (2): ESI-MS (negative ion mode) m/z 608.0820[MH] ^- , ESI-MS (positive ion mode) m/z 611.1618[M+H] ⁺ . ¹ H-NMR (400MHz, CD ₃ OD): δ _H 6.22(1H,d,H-6),6.41(1H,d,H-8),7.67(1H,d,H-2'),6.88( 1H,d,H-5'),7.63(1H,dd,H-6'),5.11(1H,d,H-1”),3.26-3.48(4H,m,H-2”,H-3 ”,H-4”,H-5”),3.39(1H,m,Ha-6”),4.52(1H,d,H-1”’),3.63(1H,dd,H-2”’) ,3.54(1H,dd,H-3'"),3.27(1H,m,H-4"'),3.44(1H,m,H-5"'),3.81(1H,dt,Hb-6" ). ¹³ C-NMR (CD ₃ OD, 100MHz): δ _C 158.69 (C-2), 136.72 (C-3), 179.61 (C-4), 162.17 (C-5), 100.10 (C-6) ,167.19(C-7),95.01(C-8),159.51(C-9),106.06(C-10),123.30(C-1'),117.84(C-2'),146.02(C-3 '),150.45(C-4'),116.22(C-5'),123.70(C-6'),104.85(C-1”),75.90(C-2”),77.41(C-3”) ,71.57(C-4”),78.36(C-5”),68.72(C-6”),102.58(C-1”’),72.27(C-2”’),72.41(C-3’” ),74.10(C-4”’),70.59(C-5”’),18.03(C-6”’)[32].

Brassin (3): ESI-MS (positive ion mode) m/z 773.0314[M+H] ⁺ , ¹ H-NMR (400MHz, DMSO-d ₆ ): δ _H 7.08 (1H,m,arom,7 -H),4.95(1H,dd,H-4a),4.05(1H,dd,H-4),4.01(3H,s,H-12),3.80(2H,d,H-11),3.76( 2H,m),3.66(1H,m,H-2),3.45(1H,dd,H-3). ¹³ C-NMR(100MHz DMSO-d ₆ ): δ _C 164.4(C-6),150.9( C-8),148.0(C-10),140.9(C-9),118.0(C-6a),115.9(C-10a),109.7(C-7),81.6(C-2),80.0(C -4a),74.2(C-4),72.9(C-10b),70.5(C-3),61.3(C-11),59.5(C-12)[33].

Kaempferol (4): ESI-MS (negative ion mode) m/z 285.2310[MH] ^- . ¹ H-NMR (400MHz, DMSO): δ _H 6.19 (1H, d, arom, H-6), 6.44 (1H ,d,arom,H-8),8.04(1H,d,arom,H-2',6'),6.92(1H,d,arom,H-3',5'). ¹³ C-NMR(100MHz DMSO): δ _C 146.9(C-2),135.7(C-3),176.0(C-4),160.8(C-5),98.3(C-6),164.0(C-7),93.6(C -8),156.3(C-9),103.1(C-10),121.8(C-1′),129.6(C-2',6'),115.5(C-3',5'),159.3( C-4')[34].

Palmitic acid (5): ESI-MS (positive ion mode) m/z 256.4321[M+H] ⁺ . ¹ H-NMR (400MHz, DMSO-d ₆ ): δ _H 0.93 (3H,t,H-16) ,1.25(24H,m,H-4to H-15),1.50(2H,H-3),2.20(2H,H-2). ¹³ C-NMR(100MHz DMSO-d ₆ ): δ _C 174.5(C -1),33.7(C-2),31.3-24.5(5signals C-3 to C-14),22.1(C-15),13.9(C-16)[32].

The present invention establishes an effective one-step internal circulation CCC method to isolate compounds from Pangasius rhizome in Pakistan. Quercetin-rhamnoside (1) and quercetin-3 were successfully separated using an optimized biphasic solvent system (TBME/n-butanol/acetonitrile/water (2:2:1:5, v/v) Five compounds -O-rhamnoside (2), fucocyanin (3), kaempferol (4) and palmitic acid (5), the purity is >98% (as shown in Figure 1). The established one-step The cyclic CCC method is suitable for the separation of compounds with similar K _D values, and has the advantages of less solvent consumption, short time consumption, and a high theoretical plate number N. The method is also suitable for the separation of compounds with similar K _D values from other naturally derived products. compound.

The above-mentioned specific embodiments are only specific cases of the present invention. The patent protection scope of the present invention includes but is not limited to the above-mentioned specific embodiments, any appropriate changes made to them by those of ordinary skill in the technical field that are consistent with the claims of the present invention. or modifications shall fall within the scope of patent protection of the present invention.

Claims (10)

1. A preparation method of bergenia plant active ingredient is characterized in that: comprising the following steps:

step 1, preparing a crude extract: extracting rhizoma corydalis Decumbentis with methanol, mixing the extractive solutions, and concentrating to obtain crude methanol extract of rhizoma corydalis Decumbentis;

step 2, selecting a biphasic solvent system: taking a methanol crude extract samplePlacing into a container, adding balanced biphasic solvent, repeatedly oscillating to make the sample completely distributed in two phases, standing to separate into two layers, and adding methanol into each layer for dilution; by bringing the lower phase into stationary phase A _L Dividing the peak area of the target compound by the phase-mobile phase A _U Calculation of K by peak area _D Value: k (K) _D ＝A _L /A _U ；

Step 3, solvent System and sample solution preparation

Hscc separation preparation: adding a biphasic solvent system containing TBME-n-butanol-acetonitrile-water into a separating funnel, vigorously oscillating, standing until the two phases are in a balanced state, wherein an upper phase and a lower phase are respectively used as a stationary phase and a mobile phase;

sample solution preparation process: adding the radix Notoginseng extract into a container, and respectively adding upper phase and lower phase of TMBE-n-butanol-acetonitrile-water solvent system to dissolve the sample completely;

step 4, high-speed countercurrent chromatographic separation

One-dimensional separation: a six-way valve is adopted, a first six-way valve is arranged between the solvent bottle and the pump, and a second six-way valve is connected between the first six-way valve and the detector; the internal circulation is controlled by a six-way valve I;

and (3) internal circulation separation: closing a six-way valve collection mode to enter an internal circulation countercurrent chromatography CCC separation process; and closing the six-way valve, returning the eluent pump to the column to form a cycle, overlapping the tail end peak of the first peak and the tail end peak of the other peak in one cycle, converting the cycle mode into a normal elution method, and adopting a sequential elution method from beginning to end to avoid overlapping peak tops so as to obtain the compound.

2. The method for preparing an active ingredient of bergenia plants according to claim 1, wherein: in the step 1, extracting the rhizome of the radix cynanchi with methanol for three times, combining the extracting solutions, and concentrating under reduced pressure at 45 ℃ to obtain a crude methanol extract of the radix cynanchi.

3. The method for preparing an active ingredient of bergenia plants according to claim 1, wherein: in the step 3, the volume ratio of TBME-n-butanol-acetonitrile-water is (1-5): (1-5): (0.5-3):

(2-10)。

4. the method for preparing an active ingredient of bergenia plants according to claim 1, wherein: in the step 3, the volume ratio of TBME-n-butanol-acetonitrile-water is 2:2:1:5.

5. The method for preparing an active ingredient of bergenia plants according to claim 1, wherein: in the step 3, the volume ratio of TBME-n-butanol-acetonitrile-water is 3:1:1:5 or 1:3:1:5.

6. the method for preparing an active ingredient of bergenia plants according to claim 1, wherein: in step 4, one-dimensional separation: pumping the upper phase-stationary phase into CCC in 30mL/min head-tail elution mode, adjusting CCC instrument rotation speed to 800 rpm at 25deg.C, pumping the lower phase-mobile phase at 2.0mL/min until equilibrium, thereby obtaining proper stationary phase retention value S _f A value; sampling after balancing, and monitoring effluent liquid at 254nm wavelength by using a portable recorder; the internal circulation valve was closed prior to elution.

7. The method for preparing an active ingredient of bergenia plants according to claim 1, wherein: the purified product comprises quercetin-rhamnoside, quercetin-3-O-rhamnoside, bergenin, kaempferol and palmitic acid.

8. The method for preparing an active ingredient of bergenia plants according to claim 1, wherein: after sample injection, switching to a circulation mode through a six-way valve, switching to a normal mode when a target peak is separated, collecting, switching to the circulation mode after collection is finished, carrying out internal circulation separation of other components, and repeating the steps to finish compound separation; the compounds kaempferol and palmitic acid are eluted in the first circulation, the compounds bergenin is eluted in the second circulation, and the compounds quercetin-rhamnoside and quercetin-3-O-rhamnoside are eluted in the fourth circulation.

9. A process for the preparation of bergenia plant active ingredient according to any one of claims 1 to 8, characterized in that: an internal circulation CCC system is adopted, and comprises a six-way valve I, a six-way valve II, a pump, a solvent bottle, a sample injector, a countercurrent separation column and a detector; the first six-way valve is connected between the solvent bottle and the pump so as to be convenient for switching between internal circulation and collection, and the second six-way valve is connected between the first six-way valve and the detector; the pump is connected with the sample injector, the sample injector is connected with the countercurrent separation column, the countercurrent separation column is connected with the detector, the detector is connected with the six-way valve II, the six-way valve II is connected with the collecting ring, and the six-way valve I is connected with the distillate pipe.

10. Use of a compound extracted by the method for preparing an active ingredient of bergenia plants according to any one of claims 1 to 8 in the pharmaceutical field.