Isolation and structural identification of chemical components of standard decoction of Rhizoma Chuanxiong

Abstract

The present invention provides a method for separating the chemical components of a standard decoction of Caulis Scutellariae, comprising the following steps: obtaining an extract of the standard decoction of Caulis Scutellariae; concentrating the extract under reduced pressure, and then performing a first reverse phase chromatography, collecting fractions by a first segmentation, and preparing Fr.1 to Fr.4; performing a second reverse phase chromatography on Fr.1, collecting fractions by a second segmentation, and preparing Fr.1a to Fr.1f; performing a third reverse phase chromatography on Fr.4, collecting fractions by a third segmentation, and preparing Fr.4a to Fr.4f; performing a first semi-preparative high performance liquid chromatography separation on Fr.1a, and preparing compound 1; performing a second semi-preparative high performance liquid chromatography separation on Fr.1b, and preparing compound 2; performing a third semi-preparative high performance liquid chromatography separation on Fr.1c, and preparing compound 3; performing a fourth semi-preparative high performance liquid chromatography separation on Fr.4d, and preparing compounds 4 and 5.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis and drug quality control, and in particular to a method for separating the chemical components of a standard decoction of Caulis Smilax china. Background Art

[0002] The Chinese herbal medicine Sichuan Akebia is the dried vine stem of the Ranunculaceae plant Clematis armandii Franch. or Clematismontana Buch.-Ham. It has the effects of diuresis, stranguria, heart-clearing and relieving vexation, and menstruation and lactation. Sichuan Akebia is mainly produced in Sichuan, Guizhou, Yunnan and Tibet, with abundant wild resources, and is a commonly used Chinese herbal medicine.

[0003] Sichuan Atractylodes has high medicinal value and development value and low toxicity, but due to long-term medication habits and medicinal resources in the medicinal material market, it is unclear in actual production and clinical medication, and the phenomenon of mixing is serious. At present, there are few reports on the chemical components of Sichuan Atractylodes, most of which are concentrated on triterpenoid saponins, flavonoids and sterols. In fact, lignans are also one of the main effective material bases of Sichuan Atractylodes. There are no reports on the separation, structural identification and pharmacological activity of the chemical components of Sichuan Atractylodes lignans. Summary of the invention

[0004] Based on this, the present invention provides a method for separating the chemical components of a standard decoction of Caulis Sinensis, and successfully separates and purifies 5 compounds from the standard decoction of Caulis Sinensis. The compound content is relatively high and all of them are separated from Caulis Sinensis for the first time.

[0005] The present invention is achieved through the following technical solutions.

[0006] A method for separating the chemical components of a standard decoction of Caulis Atractylodes lancea comprises the following steps:

[0007] Obtaining an extract of a standardized decoction of Achyranthes bidentata;

[0008] The extract is subjected to a first reverse phase chromatography, and fractions are collected in a first segmentation to prepare a first fraction Fr.1, a second fraction Fr.2, a third fraction Fr.3 and a fourth fraction Fr.4; wherein the conditions of the first reverse phase chromatography include: the eluent is methanol and water, gradient elution is adopted, and the volume percentage of methanol changes from 10% to 100%;

[0009] Fr.1 is subjected to a second reverse phase chromatography, and fractions are collected in segments for the second time to prepare a first sub-fraction Fr.1a, a second sub-fraction Fr.1b, a third sub-fraction Fr.1c, a fourth sub-fraction Fr.1d, a fifth sub-fraction Fr.1e and a sixth sub-fraction Fr.1f; wherein the conditions of the second reverse phase chromatography include: the eluent is methanol and water, gradient elution is adopted, and the volume percentage of methanol changes from 10% to 100%;

[0010] Fr.4 is subjected to a third reverse phase chromatography, and fractions are collected in sections for the third time to prepare the seventh sub-fraction Fr.4a, the eighth sub-fraction Fr.4b, the ninth sub-fraction Fr.4c, the tenth sub-fraction Fr.4d, the eleventh sub-fraction Fr.4e and the twelfth sub-fraction Fr.4f; wherein the conditions of the third reverse phase chromatography include: the eluent is acetonitrile and water, gradient elution is adopted, and the volume percentage of acetonitrile changes from 3% to 100%;

[0011] Fr.1a was subjected to the first semi-preparative HPLC separation to prepare compound 1;

[0012] Fr.1b was subjected to a second semi-preparative HPLC separation to prepare compound 2;

[0013] Fr.1c was subjected to a third semi-preparative HPLC separation to prepare compound 3;

[0014] Fr.4d was subjected to a fourth semi-preparative HPLC separation to prepare compounds 4 and 5.

[0015] In one embodiment, the preparation of the extract of the standard decoction of Atractylodes lancea comprises the following steps:

[0016] The freeze-dried powder of the standard decoction of Caulis Atractylodes lancea is mixed with 60% to 80% methanol by mass, ultrasonicated, filtered, and the filtrate is collected and concentrated.

[0017] In one embodiment, the conditions for the first semi-preparative HPLC separation include: the eluent is a mixture of acetonitrile and water with a volume fraction of (6-10):(90-94); and the chromatographic column is an octadecylsilane bonded silica gel column.

[0018] In one embodiment, the conditions for the second semi-preparative HPLC separation include: the eluent is a mixture of acetonitrile and water with a volume fraction of (13-17):(83-87); and the chromatographic column is an octadecylsilane bonded silica gel column.

[0019] In one embodiment, the conditions for the third semi-preparative HPLC separation include: the eluent is a mixture of acetonitrile and water with a volume fraction of (18-22):(78-82); and the chromatographic column is an octadecylsilane bonded silica gel column.

[0020] In one embodiment, the conditions for the fourth semi-preparative HPLC separation include: the eluent is a mixture of acetonitrile and water with a volume fraction of (28-32):(68-72); and the chromatographic column is an octadecylsilane bonded silica gel column.

[0021] In one embodiment, the first segmented collection of fractions further includes the following steps:

[0022] The first high performance liquid chromatography analysis was performed, and the same fractions were combined to prepare the first fraction Fr.1, the second fraction Fr.2, the third fraction Fr.3 and the fourth fraction Fr.4;

[0023] Among them, the conditions for the first high performance liquid chromatography analysis include: mobile phase A is acetonitrile, mobile phase B is water; gradient elution is adopted; the program of the gradient elution is: 0min~5min, the volume percentage of mobile phase A is 3%; 5min~20min, the volume percentage of mobile phase A changes from 3% to 18%; 20min~30min, the volume percentage of mobile phase A changes from 18% to 22%; 30min~60min, the volume percentage of mobile phase A changes from 22% to 35%; the chromatographic column is an octadecylsilane bonded silica gel column; the column temperature is 25℃~35℃; the flow rate is 0.5mL / min~1.5mL / min; the detection wavelength is 200nm~210nm.

[0024] In one embodiment, the second segmented collection of the flow fractions further includes the following steps:

[0025] The same fractions were combined by a second HPLC analysis to prepare the first sub-fraction Fr.1a, the second sub-fraction Fr.1b, the third sub-fraction Fr.1c, the fourth sub-fraction Fr.1d, the fifth sub-fraction Fr.1e and the sixth sub-fraction Fr.1f;

[0026] Among them, the conditions for the second high performance liquid chromatography analysis include: mobile phase A is acetonitrile, mobile phase B is water; gradient elution is adopted; the program of the gradient elution is: 0min~5min, the volume percentage of mobile phase A is 3%; 5min~20min, the volume percentage of mobile phase A changes from 3% to 18%; 20min~30min, the volume percentage of mobile phase A changes from 18% to 22%; 30min~60min, the volume percentage of mobile phase A changes from 22% to 35%; the chromatographic column is an octadecylsilane bonded silica gel column; the column temperature is 25℃~35℃; the flow rate is 0.5mL / min~1.5mL / min; the detection wavelength is 200nm~210nm.

[0027] In one embodiment, the third segmented collection of fractions further includes the following steps:

[0028] The third HPLC analysis was performed, and the same fractions were combined to prepare the seventh sub-fraction Fr.4a, the eighth sub-fraction Fr.4b, the ninth sub-fraction Fr.4c, the tenth sub-fraction Fr.4d, the eleventh sub-fraction Fr.4e and the twelfth sub-fraction Fr.4f;

[0029] Among them, the conditions for the third high performance liquid chromatography analysis include: mobile phase A is acetonitrile, mobile phase B is water; gradient elution is adopted; the program of the gradient elution is: 0min~5min, the volume percentage of mobile phase A is 3%; 5min~20min, the volume percentage of mobile phase A changes from 3% to 18%; 20min~30min, the volume percentage of mobile phase A changes from 18% to 22%; 30min~60min, the volume percentage of mobile phase A changes from 22% to 35%; the chromatographic column is an octadecylsilane bonded silica gel column; the column temperature is 25℃~35℃; the flow rate is 0.5mL / min~1.5mL / min; the detection wavelength is 200nm~210nm.

[0030] The present invention also provides a method for structural identification of chemical components of a standard decoction of Caulis Smilax china, comprising the following steps:

[0031] Obtaining chemical components separated by the chemical component separation method of the standard decoction of Caulis Smilax china as described above; the chemical components include one or more of the compound 1, compound 2, compound 3, compound 4 and compound 5;

[0032] Determine the chemical components by high performance liquid chromatography coupled with mass spectrometry;

[0033] Among them, the conditions of the high performance liquid chromatography include: mobile phase A is acetonitrile, mobile phase B is water; gradient elution is adopted; the program of the gradient elution is: 0min~0.5min, the volume percentage of mobile phase A is 3%; 0.5min~3min, the volume percentage of mobile phase A changes from 3% to 18%; 3min~5min, the volume percentage of mobile phase A changes from 18% to 22%; 5min~12min, the volume percentage of mobile phase A changes from 22% to 35%; the chromatographic column is an octadecylsilane bonded silica gel column; the column temperature is 25℃~35℃; the flow rate is 0.3mL / min~0.5mL / min; full wave scanning is performed in the range of 200nm~400nm.

[0034] In one embodiment, the mass spectrometry conditions include: using an electrospray ion source; scanning in negative ion mode; a capillary voltage of 0.7 kV to 0.9 kV; a cone voltage of 12 V to 18 V; a desolvation temperature of 420°C to 480°C; an ion source temperature of 120°C to 180°C; a scanning range of m / z100 to m / z1250; and a sampling frequency of 8 Hz to 12 Hz.

[0035] In one embodiment, the compound 1 is adenosine;

[0036] The compound 2 is larch resin alcohol-4,4′-di-O-β-D-pyranoglucoside;

[0037] The compound 3 is larch resin alcohol-4-O-β-D-pyranoglucoside;

[0038] The compound 4 is secoisolariciresinol;

[0039] The compound 5 is larch resin alcohol.

[0040] Compared with the prior art, the method for separating the chemical components of the standard decoction of Caulis Smilax china of the present invention has the following beneficial effects:

[0041] The present invention repeatedly uses reverse phase chromatography and semi-preparative high performance liquid chromatography separation technology, and limits the elution conditions of each reverse phase chromatography, and successfully separates and purifies 5 water-soluble compounds from the standard decoction of Chuanmutong. The biological activity is basically clear, the content is high, and all of them are separated from Chuanmutong for the first time. This provides a basis for in-depth research on the chemical components of the standard decoction of Chuanmutong, and further provides a useful reference for improving the quality standards of Chuanmutong medicinal materials, decoction pieces and preparations thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The ESI-MS spectrum of compound 1 provided by the present invention;

[0043] Figure 2 Compound 1 provided by the present invention is subjected to DMSO-d6 (400MHz) conditions. 1 H NMR spectrum;

[0044] Figure 3 Compound 1 provided by the present invention is subjected to DMSO-d6 (100MHz) conditions. 13 C NMR spectrum;

[0045] Figure 4 Compound 1 provided by the present invention is subjected to DMSO-d6 (100MHz) conditions. 13 C NMR and DEPT-135 spectra;

[0046] Figure 5 The ESI-MS spectrum of compound 2 provided by the present invention;

[0047] Figure 6 Compound 2 provided by the present invention is subjected to DMSO-d6 (400MHz) conditions. 1 H NMR spectrum;

[0048] Figure 7 Compound 2 provided by the present invention is subjected to DMSO-d6 (100 MHz) conditions. 13 C NMR spectrum;

[0049] Figure 8 Compound 2 provided by the present invention is subjected to DMSO-d6 (100 MHz) conditions. 13 C NMR and DEPT-135 spectra;

[0050] Fig. 9 The ESI-MS spectrum of compound 3 provided by the present invention;

[0051] Fig.10 Compound 3 provided by the present invention is subjected to DMSO-d6 (400MHz) conditions. 1 H NMR spectrum;

[0052] Fig.11 Compound 3 provided by the present invention is subjected to DMSO-d6 (100MHz) conditions. 13 C NMR spectrum;

[0053] Fig.12 Compound 3 provided by the present invention is subjected to DMSO-d6 (100MHz) conditions. 13 C NMR and DEPT-135 spectra;

[0054] Fig.13 The ESI-MS spectrum of compound 4 provided by the present invention;

[0055] Fig.14 Compound 4 provided by the present invention is subjected to DMSO-d6 (400MHz) conditions. 1 H NMR spectrum;

[0056] Fig.15 Compound 4 provided by the present invention is subjected to DMSO-d6 (100 MHz) conditions. 13 C NMR spectrum;

[0057] Fig.16 Compound 4 provided by the present invention is subjected to DMSO-d6 (100 MHz) conditions. 13C NMR and DEPT-135 spectra;

[0058] Fig.17 The ESI-MS spectrum of compound 5 provided by the present invention;

[0059] Fig.18 Compound 5 provided by the present invention is subjected to DMSO-d6 (400MHz) conditions. 1 H NMR spectrum;

[0060] Fig.19 The compound 5 provided by the present invention is subjected to DMSO-d6 (100MHz) conditions. 13 C NMR spectrum;

[0061] Fig. 20 The compound 5 provided by the present invention is subjected to DMSO-d6 (100MHz) conditions. 13 C NMR and DEPT-135 spectra. DETAILED DESCRIPTION

[0062] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0063] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the invention, the meaning of "multiple" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined. In the description of the present invention, the meaning of "several" is at least one, such as one, two, etc., unless otherwise clearly and specifically defined.

[0064] The words "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that may provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0065] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0066] Unless otherwise indicated, all molecular weights herein are weight average molecular weight expressed in Daltons.

[0067] Unless otherwise specified, all formulations and tests herein took place at 25°C.

[0068] As used herein, "includes," "comprising," "including," "containing," "having," or other variations are intended to encompass non-closed inclusions, and no distinction is made between these terms. The term "comprising" means that other steps and ingredients that do not affect the end result may be added. The term "comprising" also includes the terms "consisting of" and "consisting essentially of." The compositions and methods / processes of the present invention comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, steps, or limitations described herein. As used herein, no distinction is made between the terms "efficacy," "performance," "effect," and "efficacy."

[0069] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0070] The present invention provides a method for separating the chemical components of a standard decoction of Caulis Smilax china, comprising the following steps:

[0071] Obtaining an extract of a standardized decoction of Achyranthes bidentata;

[0072] The extract is subjected to a first reverse phase chromatography, and the fractions are collected in a first segmentation to prepare a first fraction Fr.1, a second fraction Fr.2, a third fraction Fr.3 and a fourth fraction Fr.4; wherein the conditions of the first reverse phase chromatography include: the eluent is methanol and water, and gradient elution is adopted, and the volume percentage of methanol changes from 10% to 100%;

[0073] Fr.1 is subjected to a second reverse phase chromatography, and fractions are collected in segments for the second time to prepare a first sub-fraction Fr.1a, a second sub-fraction Fr.1b, a third sub-fraction Fr.1c, a fourth sub-fraction Fr.1d, a fifth sub-fraction Fr.1e and a sixth sub-fraction Fr.1f; wherein the conditions of the second reverse phase chromatography include: the eluent is methanol and water, gradient elution is adopted, and the volume percentage of methanol changes from 10% to 100%;

[0074] Fr.4 is subjected to a third reverse phase chromatography, and fractions are collected in sections for the third time to prepare the seventh sub-fraction Fr.4a, the eighth sub-fraction Fr.4b, the ninth sub-fraction Fr.4c, the tenth sub-fraction Fr.4d, the eleventh sub-fraction Fr.4e and the twelfth sub-fraction Fr.4f; wherein the conditions of the third reverse phase chromatography include: the eluent is acetonitrile and water, gradient elution is adopted, and the volume percentage of acetonitrile changes from 3% to 100%;

[0075] Fr.1a was subjected to the first semi-preparative HPLC separation to prepare compound 1;

[0076] Fr.1b was subjected to a second semi-preparative HPLC separation to prepare compound 2;

[0077] Fr.1c was subjected to a third semi-preparative HPLC separation to prepare compound 3;

[0078] Fr.4d was subjected to a fourth semi-preparative HPLC separation to prepare compounds 4 and 5.

[0079] In a specific example, the preparation of the extract of the standard decoction of Caulis Atractylodes lancea comprises the following steps:

[0080] The freeze-dried powder of the standard decoction of Caulis Atractylodes lancea is mixed with 60% to 80% methanol by mass, ultrasonicated, filtered, and the filtrate is collected and concentrated.

[0081] More specifically, the preparation of the extract of the standard decoction of Caulis Atractylodes lancea comprises the following steps:

[0082] The freeze-dried powder of the standard decoction of Caulis Atractylodes was mixed with 70% methanol by mass, ultrasonicated, and then filtered. The filtrate was collected and concentrated under reduced pressure.

[0083] More specifically, the preparation of the extract of the standard decoction of Caulis Atractylodes lancea comprises the following steps:

[0084] Take 10g of freeze-dried powder of standard decoction of Caulis Atractylodes lancea, dissolve it with 1000ml of 70% methanol by ultrasonic, filter it, take the filtrate, and concentrate it under reduced pressure to obtain 70% extract fraction (CM fraction).

[0085] In a specific example, the preparation of the freeze-dried powder of the standard decoction of Caulis Atractylodes macrocephala slices comprises the following steps:

[0086] Take 200g of Sichuan Atractylodes slices, add water and decoct twice, add 10 times the amount of water for the first time, soak for 30 minutes, decoct for 30 minutes, filter while hot, and cool the filtrate quickly. Add 8 times the amount of water for the second time, decoct for 25 minutes, filter while hot, cool the filtrate quickly, combine the two filtrates, concentrate under reduced pressure, and freeze-dry.

[0087] In a specific example, the conditions for the first semi-preparative HPLC separation include: the eluent is a mixture of acetonitrile and water with a volume fraction of (6-10):(90-94); and the chromatographic column is an octadecylsilane bonded silica gel column.

[0088] More specifically, the eluent used in the first semi-preparative HPLC separation is a mixture of acetonitrile and water with a volume fraction of 8:92.

[0089] More specifically, the chromatographic column used for the first semi-preparative HPLC separation was Waters Xselect HSST3 C18 (4.6 mm×250 mm, 5 μm).

[0090] In a specific example, the conditions for the second semi-preparative HPLC separation include: the eluent is a mixture of acetonitrile and water with a volume fraction of (13-17):(83-87); and the chromatographic column is an octadecylsilane bonded silica gel column.

[0091] More specifically, the eluent used in the second semi-preparative HPLC separation is a mixture of acetonitrile and water with a volume fraction of 15:85.

[0092] More specifically, the chromatographic column used in the second semi-preparative HPLC separation is Waters Xselect HSST3 C18 (4.6 mm×250 mm, 5 μm).

[0093] In a specific example, the conditions for the third semi-preparative HPLC separation include: the eluent is a mixture of acetonitrile and water with a volume fraction of (18-22):(78-82); and the chromatographic column is an octadecylsilane bonded silica gel column.

[0094] More specifically, the eluent used in the third semi-preparative HPLC separation is a mixture of acetonitrile and water with a volume fraction of 20:80.

[0095] More specifically, the chromatographic column used in the third semi-preparative HPLC separation was Waters Xselect HSST3 C18 (4.6 mm×250 mm, 5 μm).

[0096] In a specific example, the conditions for the fourth semi-preparative HPLC separation include: the eluent is a mixture of acetonitrile and water with a volume fraction of (28-32):(68-72); and the chromatographic column is an octadecylsilane bonded silica gel column.

[0097] More specifically, the eluent used in the fourth semi-preparative HPLC separation is a mixture of acetonitrile and water with a volume fraction of 30:70.

[0098] More specifically, the chromatographic column used in the fourth semi-preparative HPLC separation was Waters Xselect HSST3 C18 (4.6 mm×250 mm, 5 μm).

[0099] In a specific example, the conditions for the first semi-preparative HPLC separation also include: a flow rate of 2 ml / min; and / or

[0100] The conditions for the second semi-preparative HPLC separation also include: a flow rate of 2 ml / min; and / or

[0101] The conditions for the third semi-preparative HPLC separation also include: a flow rate of 2 ml / min; and / or

[0102] The conditions for the fourth semi-preparative HPLC separation also include: a flow rate of 2 ml / min.

[0103] In a specific example, the first segmented collection of flow fractions further includes the following steps:

[0104] The first high performance liquid chromatography analysis was performed, and the same fractions were combined to prepare the first fraction Fr.1, the second fraction Fr.2, the third fraction Fr.3 and the fourth fraction Fr.4;

[0105] Among them, the conditions for the first high performance liquid chromatography analysis include: mobile phase A is acetonitrile, mobile phase B is water; gradient elution is adopted; the program of gradient elution is: 0min~5min, the volume percentage of mobile phase A is 3%; 5min~20min, the volume percentage of mobile phase A changes from 3% to 18%; 20min~30min, the volume percentage of mobile phase A changes from 18% to 22%; 30min~60min, the volume percentage of mobile phase A changes from 22% to 35%; the chromatographic column is an octadecylsilane bonded silica gel column; the column temperature is 25℃~35℃; the flow rate is 0.5mL / min~1.5mL / min; the detection wavelength is 200nm~210nm.

[0106] More specifically, the conditions for the first HPLC analysis include: column temperature of 30° C.; flow rate of 1 mL / min; detection wavelength of 205 nm; and injection volume of 10 μL.

[0107] In a specific example, the second segmented collection of flow fractions further includes the following steps:

[0108] The same fractions were combined by a second HPLC analysis to prepare the first sub-fraction Fr.1a, the second sub-fraction Fr.1b, the third sub-fraction Fr.1c, the fourth sub-fraction Fr.1d, the fifth sub-fraction Fr.1e and the sixth sub-fraction Fr.1f;

[0109] Among them, the conditions for the second high performance liquid chromatography analysis include: mobile phase A is acetonitrile, mobile phase B is water; gradient elution is adopted; the program of gradient elution is: 0min~5min, the volume percentage of mobile phase A is 3%; 5min~20min, the volume percentage of mobile phase A changes from 3% to 18%; 20min~30min, the volume percentage of mobile phase A changes from 18% to 22%; 30min~60min, the volume percentage of mobile phase A changes from 22% to 35%; the chromatographic column is octadecylsilane bonded silica gel column; the column temperature is 25℃~35℃; the flow rate is 0.5mL / min~1.5mL / min; the detection wavelength is 200nm~210nm.

[0110] More specifically, the conditions for the second HPLC analysis include: column temperature of 30° C.; flow rate of 1 mL / min; detection wavelength of 205 nm; and injection volume of 10 μL.

[0111] In a specific example, the third segmented collection of flow fractions further includes the following steps:

[0112] The third HPLC analysis was performed, and the same fractions were combined to prepare the seventh sub-fraction Fr.4a, the eighth sub-fraction Fr.4b, the ninth sub-fraction Fr.4c, the tenth sub-fraction Fr.4d, the eleventh sub-fraction Fr.4e and the twelfth sub-fraction Fr.4f;

[0113] Among them, the conditions for the third high performance liquid chromatography analysis include: mobile phase A is acetonitrile, mobile phase B is water; gradient elution is adopted; the program of gradient elution is: 0min~5min, the volume percentage of mobile phase A is 3%; 5min~20min, the volume percentage of mobile phase A changes from 3% to 18%; 20min~30min, the volume percentage of mobile phase A changes from 18% to 22%; 30min~60min, the volume percentage of mobile phase A changes from 22% to 35%; the chromatographic column is octadecylsilane bonded silica gel column; the column temperature is 25℃~35℃; the flow rate is 0.5mL / min~1.5mL / min; the detection wavelength is 200nm~210nm.

[0114] More specifically, the conditions for the third HPLC analysis include: column temperature of 30° C.; flow rate of 1 mL / min; detection wavelength of 205 nm; and injection volume of 10 μL.

[0115] The present invention also provides a method for structural identification of chemical components of a standard decoction of Caulis Smilax china, comprising the following steps:

[0116] The chemical components separated by the separation method of the chemical components of the above-mentioned standard decoction of Caulis Smilax china; the chemical components include one or more of compound 1, compound 2, compound 3, compound 4 and compound 5;

[0117] The chemical components were determined by high performance liquid chromatography coupled to mass spectrometry;

[0118] Among them, the conditions of high performance liquid chromatography include: mobile phase A is acetonitrile, mobile phase B is water; gradient elution is adopted; the program of gradient elution is: 0min~0.5min, the volume percentage of mobile phase A is 3%; 0.5min~3min, the volume percentage of mobile phase A changes from 3% to 18%; 3min~5min, the volume percentage of mobile phase A changes from 18% to 22%; 5min~12min, the volume percentage of mobile phase A changes from 22% to 35%; the chromatographic column is an octadecylsilane bonded silica gel column; the column temperature is 25℃~35℃; the flow rate is 0.3mL / min~0.5mL / min; full wave scanning is performed in the range of 200nm~400nm.

[0119] More specifically, the conditions of HPLC include: column temperature of 30° C.; flow rate of 0.4 mL / min; and injection volume of 2 μL.

[0120] More specifically, the chromatographic column was Waters Xselect HSS T3 C18 (2.1 mm×100 mm, 2.5 μm).

[0121] In a specific example, the mass spectrometry conditions include: using an electrospray ion source; scanning in negative ion mode; a capillary voltage of 0.7 kV to 0.9 kV; a cone voltage of 12 V to 18 V; a desolvation temperature of 420° C. to 480° C.; an ion source temperature of 120° C. to 180° C.; a scanning range of m / z 100 to m / z 1250; and a sampling frequency of 8 Hz to 12 Hz.

[0122] More specifically, the mass spectrometry conditions include: using an electrospray ion source; using negative ion mode scanning; a capillary voltage of 0.8 kV; a cone voltage of 15 V; a desolvation temperature of 450° C.; an ion source temperature of 150° C. to 180° C.; a scanning range of m / z100 to m / z1250; and a sampling frequency of 10 Hz.

[0123] In a specific example, compound 1 is adenosine;

[0124] Compound 2 is larch resin alcohol-4,4′-di-O-β-D-pyranoglucoside;

[0125] Compound 3 is larch resin alcohol-4-O-β-D-pyranoglucoside;

[0126] Compound 4 is secoisolariciresinol;

[0127] Compound 5 is larch resin alcohol.

[0128] Chuanmutong has a diuretic effect. Studies have shown that the diuretic effect of Chuanmutong is related to the sterols, triterpenoid saponins and lignan glycosides it contains. The 2020 Chinese Pharmacopoeia stipulates in terms of functions and indications that Chuanmutong has the effects of diuresis, relieving stranguria, clearing the heart and eliminating troubles, and promoting menstruation and lactation. This shows that in addition to diuresis, the main medicinal effects of Chuanmutong should also have antibacterial, anti-inflammatory, antiviral, and anti-tumor effects. Adenosine can exert an anti-inflammatory effect by inhibiting neutrophil chemotaxis, adhesion, respiratory burst and phagocytic function, inhibiting neutrophil aggregation and damage during myocardial ischemia-reperfusion. The larch resin alcohol and secoisolarch resin phenol contained in Chuanmutong have the effects of inhibiting α-glucosidase activity, anti-tumor, and anti-depression. Larix resin alcohol-4-O-β-D-pyranoglucoside and Larix resin alcohol-4,4′-di-O-β-D-pyranoglucoside showed a certain inhibitory effect on HSV virus, indicating that adenosine and lignans are important active ingredients of Sichuan Atractylodes. The biological activities of the five compounds isolated from Sichuan Atractylodes are basically clear, with high contents and certain specificity, which can provide a useful reference for improving the quality standards of Sichuan Atractylodes medicinal materials, decoction pieces and their preparations.

[0129] The chemical component separation method and structure identification method of the standard decoction of Caulis Smilax china of the present invention are further described in detail below in conjunction with specific examples. The raw materials used in the following examples, unless otherwise specified, are all commercially available products.

[0130] Example 1

[0131] This embodiment provides a method for separating the chemical components of a standard decoction of Caulis Smilax china, which is as follows:

[0132] 1. Instruments and test drugs

[0133] Instruments: Waters Arc UHPLC-DAD-QDa coupling instrument (Waters, USA); Shimadzu LC-20AR preparative liquid phase (Shimadzu, Japan); Bruker AvanceⅢ400MHz nuclear magnetic resonance spectrometer (Bruker, Germany); ultrapure water system Milli-Q Direct (Merck, Germany); 1 / 10,000 balance ME204E (Mettler-Toledo, Switzerland); CNC ultrasonic cleaner KQ-500DE (Kunshan Ultrasonic Instrument Co., Ltd., China); chromatographic column: Waters Xselect HSS T3C18 (250mmx4.6mm, 5μm) column; reagents: methanol (chromatographic grade, Merck, Germany), acetonitrile (chromatographic grade, Merck, Germany); water is homemade ultrapure water; other reagents are of analytical grade. Test drug: Sichuan Atractylodes slices.

[0134] 2. Experimental process

[0135] 1. Preparation of freeze-dried powder of standard decoction of Caulis Atractylodes

[0136] Take 200g of Sichuan Atractylodes slices, add water and decoct twice, add 10 times the amount of water for the first time, soak for 30 minutes, decoct for 30 minutes, filter while hot, and cool the filtrate rapidly. Add 8 times the amount of water for the second time, decoct for 25 minutes, filter while hot, and cool the filtrate rapidly, combine the two filtrates, concentrate under reduced pressure, and freeze-dry to obtain the freeze-dried powder of Sichuan Atractylodes slices standard decoction.

[0137] 2. Extraction and separation of chemical components of standard decoction of Chuanmutong

[0138] Take 10g of freeze-dried powder of standard decoction of C. chuanxiong, dissolve it with 1000ml of 70% methanol by ultrasonic, filter, take the filtrate, concentrate under reduced pressure, and obtain 70% extract fraction (CM fraction). The CM fraction is subjected to ODS column chromatography, and gradient elution is performed with methanol-water (10:90→100:0, v / v), and the flow fractions are collected in sections. The segmented flow fractions are injected and analyzed by high performance liquid chromatography, and the chromatographic column is Waters Xselect HSS T3 C18 (4.6mm×250mm, 5μm), acetonitrile is used as mobile phase A, water is used as mobile phase B, and gradient elution (0~5min, 3%A; 5~20min, 3%~18%A; 20~30min, 18%~22%A; 30~60min, 22%~35%A); the detection wavelength is 205nm; the column temperature is 30℃; the flow rate is 1.0ml / min, and the injection volume is 10μl. The same fractions were combined to obtain four fractions (Fr.1 to Fr.4).

[0139] Fr.1 was separated by ODS column, gradient eluted with methanol-water (10:90→100:0, v / v), and fractions were collected in sections. The fractions were analyzed by high performance liquid chromatography. The chromatograph was Waters e2695 high performance liquid chromatograph, the column was Waters Xselect HSS T3 C18 (4.6mm×250mm, 5μm), acetonitrile was used as mobile phase A, water was used as mobile phase B, gradient elution (0~5min, 3%A; 5~20min, 3%~18%A; 20~30min, 18%~22%A; 30~60min, 22%~35%A); the detection wavelength was 205nm; the column temperature was 30℃; the flow rate was 1.0ml / min, and the injection volume was 10μl. The same fractions were combined to obtain 6 sub-fractions (Fr.1a~Fr.1f).

[0140] Fr.1a was separated and purified by semi-preparative HPLC, the chromatograph was Shimadzu LC-20AR preparative liquid phase, the chromatographic column was Waters Xselect HSS T3 C18 (4.6mm×250mm, 5μm), the eluent was acetonitrile-water, the ratio was 8:92, the flow rate was 2ml / min, and compound 1 (≤10.0mg) was obtained;

[0141] Fr.1b was separated and purified by semi-preparative HPLC, the chromatograph was Shimadzu LC-20AR preparative liquid phase, the chromatographic column was Waters Xselect HSS T3 C18 (4.6mm×250mm, 5μm), the eluent was acetonitrile-water, the ratio was 15:85, the flow rate was 2ml / min, and compound 2 (≤5.0mg) was separated;

[0142] Fr.1c was separated and purified by semi-preparative HPLC, the chromatograph was Shimadzu LC-20AR preparative liquid phase, the chromatographic column was Waters Xselect HSS T3 C18 (4.6mm×250mm, 5μm), the eluent was acetonitrile-water, the ratio was 20:80, the flow rate was 2ml / min, and compound 3 (≤5.0mg) was obtained;

[0143] Fr.4 was chromatographed on an ODS column, eluted with acetonitrile-water (3:97-100:0, v / v) gradient, and fractions were collected in sections. The fractions were analyzed by high performance liquid chromatography. The chromatograph was Waters e2695 high performance liquid chromatograph, the column was Waters Xselect HSS T3 C18 (4.6mm×250mm, 5μm), acetonitrile was used as mobile phase A, water was used as mobile phase B, gradient elution (0~5min, 3%A; 5~20min, 3%~18%A; 20~30min, 18%~22%A; 30~60min, 22%~35%A); the detection wavelength was 205nm; the column temperature was 30℃; the flow rate was 1.0ml / min, and the injection volume was 10μl. The same fractions were combined to obtain six sub-fractions (Fr.4a~Fr.4f);

[0144] Fr.4d was separated and purified by semi-preparative HPLC. The chromatograph was Shimadzu LC-20AR preparative liquid phase, the chromatographic column was Waters Xselect HSS T3 C18 (4.6mm×250mm, 5μm), the eluent was acetonitrile-water in a ratio of 30:70, the elution was isocratic, and the flow rate was 2ml / min to obtain compound 4 (≤6.0mg) and compound 5 (≤5.0mg).

[0145] Example 2

[0146] This embodiment provides a method for structural identification of the chemical components of a standard decoction of Caulis Smilax china, which is as follows:

[0147] 1. Experimental process

[0148] A 400 MHz nuclear magnetic resonance spectrometer was used to collect hydrogen and carbon spectra of the samples, and the sample solvent was DMSO-d6. A Waters Arc UHPLC-DAD-QDa liquid chromatography-mass spectrometer was used to collect chromatograms and mass spectra of the samples. The chromatographic column was Waters Xselect HSS T3 C18 (2.1mm×100mm, 2.5μm), with acetonitrile as mobile phase A, water as mobile phase B, and gradient elution (0-0.5min, 3%A; 0.5-3min, 3%-18%A; 3-5min, 18%-22%A; 5-12min, 22%-35%A); full wave scanning was performed in the range of 200nm-400nm, the column temperature was 30℃; the flow rate was 0.4ml / min, and the injection volume was 2μl; mass spectrometry ion source: electrospray ion source (ESI); negative ion mode scanning; capillary voltage was 0.8kv, cone voltage was 15v, desolvation temperature was 450℃, ion source temperature was 150℃, scanning range was m / z100-m / z1250, sampling frequency was 10Hz, and the structures of compounds 1-5 were identified.

[0149] The structural identification spectra of compounds 1 to 5 are shown in Figures 1 to 20 shown.

[0150] 2. Structural Identification Results

[0151] 1. Compound 1

[0152]

[0153] Pale yellow powder (methanol-water); ESI-MS m / z: 302 [M+Cl]-. 1 H-NMR(400MHz,DMSO-d6)δ:8.35(1H,s,H-8),8.14(1H,s,H-2),7.37(2H,s,6-NH2),5.87(1H,d,J=6.2Hz,H-1′),5.47(2H,m,2′,5′-OH),5.2 3(1H,s,3′-OH),4.61(1H,m,H-2′),4.14(1H,s,H-3′),3.96(1H,q,J=3.4Hz,H-4′),3.68(1H,dt,J=12.1,3.9Hz,H-5′a),3.56(1H,m,H-5′b); 13 C-NMR (100 MHz, DMSO-d6) δ: 156.2 (C-6), 152.4 (C-2), 149.1 (C-4), 139.9 (C-8), 119.4 (C-5), 87.9 (C-1′), 85.9 (C-4′), 73.4 (C-2′), 70.7 (C-3′), 61.7 (C-5′). Compound 1 was identified as adenosine.

[0154] 2. Compound 2

[0155]

[0156] Pale yellow oil (methanol); ESI-MS m / z: 719 [M+Cl] - . 1H-NMR (400MHz, DMSO-d6) δ: 7.02 (1H, d, J = 8.4Hz, H-5), 6.98 (1H, d, J = 8.4Hz, H-5′), 6.8 8(1H,d,J=2.0Hz,H-2),6.82(1H,d,J=2.0Hz,H-2′),6.78(1H,dd,J=8.4,2.0Hz,H-6),6 .68(1H,dd,J=8.3,2.0Hz,H-6′),4.87(1H,d,J=7.0Hz,H-1″′),4.85(1H,d,J=7.2Hz,H- 1″),4.72(1H,d,J=6.0Hz,H-7),3.90(1H,m,H-9′b),3.74(6H,s,OMe-3,3′),3.58(1H,m H-9′a),2.85(1H,dd,J=13.4,4.7Hz,H-7′b),2.62(1H,m,H-8′),2.46(1H,overlapped,H-7′a),2.20(1H,m,H-8); 13 C-NMR(100MHz,DMSO-d6)δ:148.8(C-3),148.7(C-3′),145.5(C-4),144.8(C-4′),137.6(C-1),134.6(C -1′),120.3(C-6′),117.8(C-6),115.2(C-5′),115.0(C-5),113.0(C-2′),110.1(C-2),100.2(C-1″),1 00.1(C-1″′),81.6(C-7),77.0(C-5″,5″′),76.9(C-3″,3″′),73.2(C-2″,2″′),71.9(C-9′),69.7(C-4″,4″′),60.7(C-6″,6″′),58.6(C-9),55.6(-OCH3),55.6(-OCH3),52.5(C-8),41.8(C-8′),32.2(C-7′). Compound 2 was identified as larch resin alcohol-4,4′-di-O-β-D-pyranoglucoside (lariciresinol-4,4′-bis-O-β-D-glucopyranoside).

[0157] 3. Compound 3

[0158]

[0159] Pale yellow oil (methanol); ESI-MS m / z: 557 [M+Cl] - ,521[MH]-. 1H-NMR(400MHz,DMSO-d6)δ:7.02(1H,d,J=8.4Hz,H-5),6.88(1H,J=2.1,H-2),6.78(1H,dd,J=8.4,2.1Hz,H-6),6.74(1H,d ,J=2.0Hz,H-2′),6.67(1H,d,J=8.0Hz,H-6′),6.57(1H,dd,J=8.0,2.0Hz,H-5′),4.87(1H,d,J=7.0Hz,Glc-H-1),4.71(1H, d,J=6.0Hz,H-7),3.89(1H,m,H-9′a),3.75(3H,s,3-OCH3),3.74(3H,s,3′-OCH3),3.66(1H,m,H-9a),3.57(1H,m,H-9′b),3 .51(1H, overlapped,H-9b),2.80(1H,dd,J=13.5,4.8Hz,H-7′a),2.57(1H,m,H-8′),2.42(1H,m,H-7′b),2.19(1H,m,H-8); 13 C-NMR(100MHz,DMSO-d6)δ:148.7(C-3),147.4(C-3′),145.5(C-4),144.6(C-4′),137.7(C-1 ),131.7(C-1′),120.6(C-6′),117.7(C-6),115.4(C-5′),115.0(C-5),112.7(C-2′),110.1(C -2), 100.1(C-1″), 81.6(C-7), 77.0(C-5″), 76.9(C-3″), 73.2(C-2″), 71.9(C-9′), 69.7(C-4″), 60.7(C-6″), 58.6(C-9), 55.6(-OCH3), 55.5(-OCH3), 52.5(C-8), 41.9(C-8′), 32.1(C-7′). Compound 3 was identified as lariciresinol-4-O-β-D-glucopyranoside.

[0160] 4. Compound 4

[0161]

[0162] Pale yellow oil (methanol); ESI-MS m / z: 361 [MH] - . 1H-NMR(400MHz,DMSO-d6)δ:8.65(2H,s,4,4′-OH),6.62(2H,overlapped,H-5,5′),6.62(2H,overlapped,H-2,2′),6.49(2H,dd,J=8.0,2.0Hz,H -6,6′),4.54(2H,brs,9,9′-OH),3.67(6H,s,3,3′-OCH3),3.37(4H,overlapped,H-9,9′),2.52(4H,overlapped,H-7,7′),1.82(2H,m,H-8,8′); 13 C-NMR (100MHz, DMSO-d6) δ: 147.2 (C-3, 3′), 144.3 (C-4, 4′), 132.2 (C-1, 1′), 121.1 (C-6, 6′), 115.0 (C-5, 5′), 112.9 (C-2, 2′), 60.2 (C-9, 9′), 55.4 (3, 3′-OCH3), 42.4 (C-8, 8′), 33.9 (C-7, 7′). Compound 4 was identified as secoisolariciresinol.

[0163] 5. Compound 5

[0164]

[0165] Pale yellow oil (methanol); ESI-MS m / z: 359 [MH] - . 1H-NMR(400MHz,DMSO-d6)δ:8.82(2H,overlapped,4,4′-OH),6.82(1H,d,J=1.8Hz,H-2′),6.74(1H,d,J=1.9Hz,H-2),6.71(1H,br d,J=8.0Hz,H-6′),6.67(2H,d,J=8.0Hz,H-5,5′),6.57(1H,dd,J=7.9,2.0Hz,H-6),4.71(1H,br s,9′-OH),4.64(1H,d,J=6.3Hz,H-7′),3.87(1H,dd,J=8.2,6.5Hz,H-9a),3.74( 3H,s,3-OCH3),3.74(3H,s,3′-OCH3),3.64(1H,d,J=8.2Hz,H-9′a),3.55(1H,dd, J=8.2,6.5Hz,H-9b),3.46(1H,overlapped,H-9′b),2.82(1H,dd,J=13.4,4.7Hz, H-7a),2.58(1H,m,H-8),2.41(1H,dd,J=13.4,10.9Hz,H-7b),2.18(1H,m,H-8′); 13 C-NMR (100MHz, DMSO-d6) δ: 147.4 (C-3′), 147.4 (C-3), 145.5 (C-4′), 144.6 (C-4), 134.7 (C-1′), 131.7 (C-1), 120.6 (C-6), 118.2 (C-6′), 115.4 (C-5′), 115.0 (C-5), 112.7 (C-2), 109.9 (C-2′), 81.8 (C-7′), 71.8 (C-9), 58.6 (C-9′), 55.6 (3-OCH3), 55.5 (3′-OCH3), 52.4 (C-8′), 42.0 (C-8), 32.2 (C-7). Compound 5 was identified as lariciresinol.

[0166] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0167] The above-described embodiments only express several implementation methods of the present invention, which are convenient for understanding the technical solutions of the present invention in detail, but they cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the protection scope of the claims attached to the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the contents of the attached claims, and the description and drawings can be used to interpret the contents of the claims.

Claims

1. A method for separating the chemical components of a standard decoction of Caulis Achyranthis Bidentatae, characterized in that: The steps include: Obtaining an extract of a standardized decoction of Achyranthes bidentata; The extract is subjected to a first reverse phase chromatography, and fractions are collected in a first segmentation to prepare a first fraction Fr.1, a second fraction Fr.2, a third fraction Fr.3 and a fourth fraction Fr.4; wherein the conditions of the first reverse phase chromatography include: the eluent is methanol and water, gradient elution is used, and the volume percentage of methanol changes from 10% to 100%; Fr.1 is subjected to a second reverse phase chromatography, and fractions are collected in the second segmentation to prepare a first sub-fraction Fr.1a, a second sub-fraction Fr.1b, a third sub-fraction Fr.1c, a fourth sub-fraction Fr.1d, a fifth sub-fraction Fr.1e and a sixth sub-fraction Fr.1f; wherein the conditions of the second reverse phase chromatography include: the eluent is methanol and water, gradient elution is adopted, and the volume percentage of methanol changes from 10% to 100%; Fr.4 was subjected to a third reverse phase chromatography, and fractions were collected in sections for the third time to prepare the seventh sub-fraction Fr.4a, the eighth sub-fraction Fr.4b, the ninth sub-fraction Fr.4c, the tenth sub-fraction Fr.4d, the eleventh sub-fraction Fr.4e and the twelfth sub-fraction Fr.4f; wherein the conditions of the third reverse phase chromatography include: the eluent is acetonitrile and water, gradient elution is adopted, and the volume percentage of acetonitrile changes from 3% to 100%; Fr.1a was subjected to the first semi-preparative HPLC separation to prepare compound 1; Fr.1b was subjected to a second semi-preparative HPLC separation to prepare compound 2; Fr.1c was subjected to a third semi-preparative HPLC separation to prepare compound 3; Fr.4d was subjected to the fourth semi-preparative HPLC separation to prepare compound 4 and compound 5; The conditions for the first semi-preparative high performance liquid chromatography separation include: the eluent is a mixture of acetonitrile and water with a volume fraction of (6-10):(90-94); the chromatographic column is an octadecylsilane bonded silica gel column; The conditions for the second semi-preparative high performance liquid chromatography separation include: the eluent is a mixture of acetonitrile and water with a volume fraction of (13-17):(83-87); the chromatographic column is an octadecylsilane bonded silica gel column; The conditions for the third semi-preparative high performance liquid chromatography separation include: the eluent is a mixture of acetonitrile and water with a volume fraction of (18-22):(78-82); the chromatographic column is an octadecylsilane bonded silica gel column; The conditions for the fourth semi-preparative high performance liquid chromatography separation include: the eluent is a mixture of acetonitrile and water with a volume fraction of (28-32):(68-72); the chromatographic column is an octadecylsilane bonded silica gel column; After the first segmented collection of flow fractions, the following steps are also included: The first high performance liquid chromatography analysis was performed, and the same fractions were combined to prepare the first fraction Fr.1, the second fraction Fr.2, the third fraction Fr.3 and the fourth fraction Fr.4; The conditions for the first high performance liquid chromatography analysis include: mobile phase A is acetonitrile, mobile phase B is water; gradient elution is used; the gradient elution program is: 0min~5min, the volume percentage of mobile phase A is 3%; 5min~20min, the volume percentage of mobile phase A changes from 3% to 18%; 20min~30min, the volume percentage of mobile phase A changes from 18% to 22%; 30min~60min, the volume percentage of mobile phase A changes from 22% to 35%; the chromatographic column is an octadecylsilane bonded silica gel column; the column temperature is 25°C~35°C; the flow rate is 0.5mL / min~1.5mL / min; the detection wavelength is 200nm~210nm; The second segmentation and collection of the flow fractions also includes the following steps: The same fractions were combined by a second HPLC analysis to prepare the first sub-fraction Fr.1a, the second sub-fraction Fr.1b, the third sub-fraction Fr.1c, the fourth sub-fraction Fr.1d, the fifth sub-fraction Fr.1e and the sixth sub-fraction Fr.1f; The conditions for the second high performance liquid chromatography analysis include: mobile phase A is acetonitrile, mobile phase B is water; gradient elution is used; the gradient elution program is: 0min~5min, the volume percentage of mobile phase A is 3%; 5min~20min, the volume percentage of mobile phase A changes from 3% to 18%; 20min~30min, the volume percentage of mobile phase A changes from 18% to 22%; 30min~60min, the volume percentage of mobile phase A changes from 22% to 35%; the chromatographic column is an octadecylsilane bonded silica gel column; the column temperature is 25°C~35°C; the flow rate is 0.5mL / min~1.5mL / min; the detection wavelength is 200nm~210nm; After the third segmented collection of the flow fractions, the following steps are also included: The third HPLC analysis was performed, and the same fractions were combined to prepare the seventh sub-fraction Fr.4a, the eighth sub-fraction Fr.4b, the ninth sub-fraction Fr.4c, the tenth sub-fraction Fr.4d, the eleventh sub-fraction Fr.4e and the twelfth sub-fraction Fr.4f; The conditions for the third high performance liquid chromatography analysis include: mobile phase A is acetonitrile, mobile phase B is water; gradient elution is used; the gradient elution program is: 0min~5min, the volume percentage of mobile phase A is 3%; 5min~20min, the volume percentage of mobile phase A changes from 3% to 18%; 20min~30min, the volume percentage of mobile phase A changes from 18% to 22%; 30min~60min, the volume percentage of mobile phase A changes from 22% to 35%; the chromatographic column is an octadecylsilane bonded silica gel column; the column temperature is 25°C~35°C; the flow rate is 0.5mL / min~1.5mL / min; the detection wavelength is 200nm~210nm; The compound 1 is adenosine; The compound 2 is larch resin alcohol-4,4′-di-O-β-D-pyranoglucoside; The compound 3 is larch resin alcohol-4-O-β-D-pyranoglucoside; The compound 4 is secoisolariciresinol; The compound 5 is larch resin alcohol.

2. The method for separating the chemical components of the standard decoction of Caulis Smilax china according to claim 1, characterized in that: The preparation of the extract of the standard decoction of Caulis Atractylodes lancea comprises the following steps: The freeze-dried powder of the standard decoction of Caulis Atractylodes lancea is mixed with 60% to 80% methanol by mass, ultrasonicated, and then filtered to collect the filtrate and concentrate it.

3. The method for separating the chemical components of the standard decoction of Caulis Smilax china according to claim 1 or 2, characterized in that: The obtained compounds were determined by high performance liquid chromatography coupled with mass spectrometry; Among them, the conditions of the high performance liquid chromatography include: mobile phase A is acetonitrile, mobile phase B is water; gradient elution is adopted; the program of the gradient elution is: 0min~0.5min, the volume percentage of mobile phase A is 3%; 0.5min~3min, the volume percentage of mobile phase A changes from 3% to 18%; 3min~5min, the volume percentage of mobile phase A changes from 18% to 22%; 5min~12min, the volume percentage of mobile phase A changes from 22% to 35%; the chromatographic column is an octadecylsilane bonded silica gel column; the column temperature is 25℃~35℃; the flow rate is 0.3mL / min~0.5mL / min; full wave scanning is performed in the range of 200nm~400nm.

4. The method for separating the chemical components of the standard decoction of Caulis Smilax china according to claim 3, characterized in that: The mass spectrometry conditions include: using an electrospray ion source; using negative ion mode scanning; the capillary voltage is 0.7kV~0.9kV; the cone voltage is 12V~18V; the desolvation temperature is 420℃~480℃; the ion source temperature is 120℃~180℃; the scanning range is m / z100~m / z1250; and the sampling frequency is 8Hz~12Hz.

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