METHOD OF ESTABLISHING FINGERPRINT SPECTRUM OF TRADITIONAL CHINESE MEDICINE COMPOSITION WITH EFFECT OF REGULATING DEPRESSION EMOTION OR FORMULATION THEREOF

Abstract

The present invention relates to the field of analysis of traditional Chinese medicine, and in particular to a method of establishing a fingerprint spectrum of a traditional Chinese medicine composition with effect of regulating depression emotion or a formulation thereof, wherein the traditional Chinese medicine composition consists of the following traditional Chinese medicine extracts in parts by weight: 40˜80 parts of Hypericum perforatum extract, 10˜40 parts of Acanthopanax senticosus extract, and 2˜20 parts of tree peony root bark extract; and the method comprises establishing the fingerprint spectrum by high performance liquid chromatography, and the chromatographic conditions are as follows: an octadecylsilane bonded silica gel chromatographic column is used as a chromatographic column; column temperature is 25˜35° C.; flow rate is 0.5˜1.1 mL/min; detection wavelength is 290˜310 nm; mobile phase A is phosphoric acid aqueous solution, mobile phase B is acetonitrile, and a gradient elution program is used.

Claims

1. A method of establishing a fingerprint spectrum of a traditional Chinese medicine composition with effect of regulating depression emotion or a formulation thereof, wherein the traditional Chinese medicine composition consists of the following traditional Chinese medicine extracts in parts by weight: 40˜80 parts of Hypericum perforatum extract, 10˜40 parts of Acanthopanax senticosus extract, and 2˜20 parts of tree peony root bark extract; and the fingerprint spectrum is constructed respectively by high performance liquid chromatography for a tested solution and an reference solution and the chromatographic conditions are as follows: an octadecylsilane bonded silica gel chromatographic column is used as a chromatographic column; column temperature is 25˜35° C.; flow rate is 0.5˜1.1 mL/min; detection wavelength is 290˜310 nm; mobile phase A is 0.05%˜0.15% phosphoric acid aqueous solution, mobile phase B is acetonitrile, and gradient elution program is as follows: TABLE-US-00004 time/min A/% B/%  0 90 10  8 86 14 12 86 14 20 80 20 30 75 25 40 60 40 42 40 60 50 40 60 52 90 10 60 90 10

2. The method according to claim 1, wherein the detection wavelength is 300 nm.

3. The method according to claim 1, wherein the mobile phase A is 0.1% phosphoric acid aqueous solution.

4. The method according to claim 1, wherein the chromatographic column is Agilent TC-C18 chromatographic column of 250×4.6 mm, 5 m.

5. The method according to claim 1, wherein the column temperature is 30° C.

6. The method according to claim 1, wherein the flow rate is 1.0 mL/min.

7. The method according to claim 1, wherein the tested solution is prepared by a method comprising steps of: mixing the traditional Chinese medicine composition or the formulation thereof with 40 vt %˜60 vt % methanol aqueous solution with a ratio of the traditional Chinese medicine composition or the formulation thereof to the methanol aqueous solution in g/mL of (0.5˜2.0): 25, performing ultrasonic treatment and filtration, and collecting subsequent filtrate; and the reference solution is prepared by a method comprising steps of: mixing hyperoside reference substance or paeonol reference substance with 40 vt %˜60 vt % methanol aqueous solution with a ratio of the hyperoside reference substance or the paeonol reference substance to the methanol aqueous solution in g/mL of (0.5˜1.5):1.

8. The method according to claim 1, wherein the traditional Chinese medicine composition consists of the following traditional Chinese medicine extracts in parts by weight: 65˜80 parts of Hypericum perforatum extract, 10˜35 parts of Acanthopanax senticosus extract, and 2˜10 parts of tree peony root bark extract.

9. The method according to claim 1, wherein the traditional Chinese medicine composition is prepared by a method comprising steps of: (1) to Hypericum perforatum, adding 10˜12 times of weight of 60 vt %˜80 vt % ethanol aqueous solution containing 0.05%-2% sodium hydroxide, extracting under heating and refluxing 2 times for 1.5˜2.0 hours each time, combining the two filtrates and filtering, concentrating the filtrate to an extract with a relative density of 1.11˜1.13 at 72° C., and performing spray-drying to obtain Hypericum perforatum extract; (2) to Acanthopanax senticosus pieces, adding 8˜10 times of weight of 0 vt %˜80 vt % ethanol aqueous solution, extracting under heating and refluxing 3 times for 1.0˜2.5 hours each, combining the three filtrates and filtering, concentrating the filtrate to an extract with a relative density of 1.11˜1.13 at 72° C., and performing spray-drying to obtain Acanthopanax senticosus extract; (3) to tree peony root bark, adding 10˜14 times its weight of water, heating and recovering 8˜10 times its amount of distillate, refrigerating for 20˜24 hours, crystallizing, filtering, and low temperature drying to obtain tree peony root bark extract; and (4) mixing the Hypericum perforatum extract, the Acanthopanax senticosus extract and the tree peony root bark extract according to a formula atio.

10. The method according to claim 1, wherein the traditional Chinese medicine composition is prepared by a method comprising steps of: (1) to Hypericum perforatum, adding 10 times of weight of 80 vt % ethanol aqueous solution containing 0.10% sodium hydroxide, extracting under heating and refluxing 2 times for 1.5 hours each time, combining the two filtrates and filtering, concentrating the filtrate to an extract with a relative density of 1.12 at 72° C., and performing spray-drying to obtain Hypericum perforatum extract; (2) to Acanthopanax senticosus pieces, adding 10 times of weight of the aqueous solution, extracting under heating and refluxing 3 times for 1.0 hours each time, combining the three filtrates and filtering, concentrating the filtrate to an extract with a relative density of 1.12 at 72° C., and performing spray-drying to obtain Acanthopanax senticosus extract; (3) to tree peony root bark, adding 14 times of weight of water, heating and recovering 9 times of amount of distillate, refrigerating for 24 hours, crystallizing, filtering, and performing low temperature drying to obtain tree peony root bark extract; and (4) mixing the Hypericum perforatum extract, the Acanthopanax senticosus extract and the tree peony root bark extract according to a formula ratio.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0042] FIG. 1 shows the fingerprint spectrum of the traditional Chinese medicine composition of the present invention;

[0043] FIG. 2 shows the fingerprint spectrum of Hypericum perforatum;

[0044] FIG. 3 shows the fingerprint spectrum of Acanthopanax senticosus;

[0045] FIG. 4 shows the fingerprint spectrum of paeonol.

DETAILED DESCRIPTION

[0046] The invention discloses a method of establishing a fingerprint spectrum of a traditional Chinese medicine composition with the effect of regulating depression emotion or a formulation thereof, which can be achieved by those skilled in the art through appropriately improving the process parameters in light of the present disclosure. In particular, it should be noted that, all similar substitutions and modifications will be apparent to those skilled in the art, and are all considered to be included in the present invention. Although the method and use of the present invention have been described by means of preferred examples, it is apparent to those skilled in the art that modifications or proper alterations and combinations can be made to the method and use described herein without departing from the content, spirit and scope of the present invention, so as to achieve and apply the technique of the present invention.

[0047] The invention discloses a method of establishing a fingerprint spectrum of a traditional Chinese medicine composition with the effect of regulating depression emotion and the fingerprint spectrum, belonging to the field of the analysis of traditional Chinese medicine.

[0048] The traditional Chinese medicine composition with the effect of regulating depression emotion is prepared by the following method:

[0049] to 77% of Hypericum perforatum, 20% of Acanthopanax senticosus, and 3% of Paeonol, 50% ethanol in parts by weight are added, extracted twice, filtered, and the two filtrates are combined and concentrated to an extract with a relative density of 1.11˜1.13 at 72° C., and then maltodextrin is added, mixed, dried, and granulated.

[0050] The tested solution is prepared as follows:

[0051] the traditional Chinese medicine composition is mixed well and porphyrized, and then 0.5˜2.0 parts by weight are placed in a triangular container with a plug, and 25 parts by volume of 50% methanol are added, weighed, ultrasonically processed, cooled down, and then weighed. 50% methanol is added to make up the lost weight, shook well, filtered, and the subsequent filtrate is collected.

[0052] The reference solution is prepared as follows:

[0053] an appropriate amount of hyperoside or paeonol reference substance is weighed, and 50% methanol is added to obtain a solution containing 1 part by weight per 1 part by volume.

[0054] The invention employs high performance liquid chromatography to establish fingerprint spectrum. In addition, an octadecylsilane bonded silica gel chromatographic column is used as a chromatographic column; column temperature: 25˜35° C.; flow rate: 0.5˜1.1 mL/min; detection wavelength: 300 nm; and 0.1% phosphoric acid solution A-acetonitrile B are used as the mobile phases, and gradient elution is performed in the following order:

TABLE-US-00002 time/min 0.1% phosphoric acid/% acetonitrile/%  0 90 10  8 86 14 12 86 14 20 80 20 30 75 25 40 60 40 42 40 60 50 40 60 52 90 10 60 90 10

[0055] The fingerprint spectrum of the present invention comprehensively reflects the quality information of the traditional Chinese medicine composition, thereby achieving the purpose of more comprehensively and effectively controlling the product quality of this traditional Chinese medicine composition.

[0056] The fingerprint spectrum comprises 11 common peaks, and the relative retention time of each peak and the reference peak are respectively: 8.291±0.05 min (Peak 1), 11.481±0.05 min (Peak 2), 12.173±0.05 min (Peak 3), 13.061±0.05 min (Peak 4), 25.763±0.05 min (Peak 5), 26.717±0.05 min (Peak 6), 27.303±0.05 min (Peak 7), 30.777±0.05 min (Peak 8), 31.521±0.05 min (Peak 9), 41.037±0.05 min (Peak 10), 45.426±0.05 min (Peak 11), of which Peak 6 is hyperoside.

[0057] The reagents or instruments used in the method of establishing a fingerprint spectrum of a traditional Chinese medicine composition with the effect of regulating depression emotion or a formulation thereof provided by the present invention are all commercially available.

[0058] The present invention will be further illustrated by the following examples:

Example 1 Preparation of a Traditional Chinese Medicine Composition with the Effect of Regulating Depression Emotion

[0059] The traditional Chinese medicine formula composition for improving depression emotion and sleep involved in the present invention mainly consists of Hypericum perforatum extract, Acanthopanax senticosus extract and tree peony root bark extract. The preparation method of the composition comprises:

[0060] to Hypericum perforatum, 10 times of amount of 80% ethanol containing 0.1% sodium hydroxide was added, extracted 2 times under heating and refluxing for 1.5 hours each time. The two filtrates were combined, filtered, and concentrated to an extract with a relative density of about 1.12 (72° C.). The extract was spray-dried to obtain a dry extract powder;

[0061] after Acanthopanax senticosus medicinal material was broken into pieces, 10 times of amount of aqueous solution was added, extracted 3 times under heating and refluxing for 1.0 hour each time. Then the decoction was combined and filtered, and the filtrate was concentrated to an extract with a relative density of 1.12 (72° C.). The extract was spray-dried to obtain a dry extract powder; and

[0062] to tree peony root bark medicinal material, 14 times of amount of water was added, heated to recover 9 times of amount of distillate, refrigerated for 24 hours, crystallized, filtered, and low temperature dried. The above three extract powders were mixed according to a ratio of 77%:20%:3% of the three component Hypericum perforatum extract, Acanthopanax senticosus extract, and paeonol extract, to obtain the composition.

Example 2 Preparation of Hypericum perforatum Extract

[0063] After Hypericum perforatum medicinal material was broken into pieces, 10 times of amount of 70% ethanol was added, extracted 2 times under heating and refluxing for 1.5 hours each time. The two filtrates were combined, filtered, and concentrated to an extract with a relative density of about 1.12 (72° C.). The extract was spray-dried to obtain Hypericum perforatum extract.

Example 3 Preparation of Acanthopanax senticosus Extract

[0064] After Acanthopanax senticosus medicinal material was broken into pieces, 10 times of amount of 50% ethanol was added, extracted 3 times under heating and refluxing for 1.0 hour each time. Then the decoction was combined and filtered, and the filtrate was concentrated to an extract with a relative density of 1.12 (72° C.). The extract was spray-dried to obtain Acanthopanax senticosus extract.

Example 4 Preparation of Tree Peony Root Bark Extract

[0065] to tree peony root bark medicinal material, 14 times of amount of water was added, heated to recover 9 times of amount of distillate, refrigerated for 24 hours, crystallized, filtered, and low temperature dried to obtain tree peony root bark extract.

Example 5 Establishment of a Reference Fingerprint Spectrum of the Traditional Chinese Medicine Formula Composition for Improving Depression Emotion and Sleep

[0066] 1. Sample: A reference substance of the traditional Chinese medicine formula composition for improving depression emotion and sleep was provided by Qingdao Silver Century Health Industry Group Co., Ltd.

[0067] 2. Reagents: acetonitrile (HPLC, Wokai); methanol (HPLC, Wokai); phosphoric acid was analytically pure; and UPLC purified water was self-made by Milli-Q Pure Water System.

[0068] 3. Instrument: Agilent Infinity 1260 (CA, USA).

[0069] 4. Preparation of the reference solution of the composition: 0.71 g of the traditional Chinese medicine formula composition was placed into a 100 mL triangular conical flask, and 25 mL of 50% CH.sub.3OH was added, accurately weighed, ultrasonicated for 20 min, and cooled to room temperature before weighing. 50% CH.sub.3OH was added to make up the lost weight, shook well, filtered, and then an appropriate amount of filtrate was taken and passed through a 0.45 m microfiltration membrane to obtain the reference solution.

[0070] 5. Chromatographic conditions: Chromatographic column was Agilent TC-C18 column (250×4.6 mm 5 μm), detection wavelength was 300 nm, column temperature was 30° C., flow rate was 1.0 mL/min, and mobile phases were acetonitrile-0.1% phosphoric acid aqueous solution with 0.1% phosphoric acid aqueous solution as A and acetonitrile as B. The injection volume was 10 μL, the analysis time was 60 min, and gradient elution was performed according to the following elution program:

TABLE-US-00003 TABLE 1 elution program time/min A 0.1% phosphoric acid/% B acetonitrile/%  0 90 10  8 86 14 12 86 14 20 80 20 30 75 25 40 60 40 42 40 60 50 40 60 52 90 10 60 90 10

[0071] The fingerprint spectrum (see FIG. 1) of the traditional Chinese medicine formula composition for improving depression emotion and sleep obtained by the above high performance liquid chromatography method comprises 11 common fingerprint peaks, which are:

[0072] Peak 1 having an average retention time RT of 8.291 min, an RSD of 1.06%, and an average peak area of 1389.931, which accounts for 3.481% of the peak area;

[0073] Peak 2 having an average retention time RT of 11.481 min, an RSD of 1.43%, and an average peak area of 828.957, which accounts for 2.076% of the peak area;

[0074] Peak 3 having an average retention time RT of 12.173 min, an RSD of 0.92%, and an average peak area of 2552.112, which accounts for 6.391% of the peak area;

[0075] Peak 4 having an average retention time RT of 13.061 min, an RSD of 1.01%, and an average peak area of 1198.181, which accounts for 3.001% of the peak area;

[0076] Peak 5 having an average retention time RT of 25.763 min, an RSD of 1.23%, and an average peak area of 3074.151, which accounts for 7.698% of the peak area;

[0077] Peak 6 having an average retention time RT of 26.717 min, an RSD of 0.95%, and an average peak area of 2400.017, which accounts for 6.010% of the peak area;

[0078] Peak 7 having an average retention time RT of 27.303 min, an RSD of 0.86%, and an average peak area of 2736.155, which accounts for 6.852% of the peak area;

[0079] Peak 8 having an average retention time RT of 30.777 min, an RSD of 0.98%, and an average peak area of 1,260.393, which accounts for 3.156% of the peak area;

[0080] Peak 9 having an average retention time RT of 31.521 min, an RSD of 0.87%, and an average peak area of 811.315, which accounts for 2.032% of the peak area;

[0081] Peak 10 having an average retention time RT of 41.037 min, an RSD of 0.6%, and an average peak area of 735.617, which accounts for 1.842% of the peak area; and

[0082] Peak 11 having an average retention time RT of 45.426 min, an RSD of 0.5%, and an average peak area of 15062.947, which accounts for 37.721% of the peak area.

[0083] 6. Identification of active ingredients of the traditional Chinese medicine formula composition for improving depression emotion and sleep

[0084] In the present invention, two target ingredients in the reference fingerprint spectrum of the traditional Chinese medicine formula composition were identified by measuring the retention time of an active ingredient reference substance. The active ingredient reference substance was determined by high performance liquid chromatography as follows:

[0085] 1) The active ingredient reference substance: hyperoside (Y04A9X62302) and paeonol (1101708-201407) were both purchased from National Institutes for Food and Drug Control.

[0086] 2) Preparation of the active ingredient reference solution: appropriate amounts of the two reference substances were taken respectively, and 50% methanol aqueous solution was added to the measuring flask, sonicated for 2 min, and diluted to the mark.

[0087] 3) Liquid chromatographic conditions and determination methods were the same as those in “(1) Determination of a reference fingerprint spectrum of the traditional Chinese medicine formula composition for improving depression emotion and sleep”.

[0088] Through the comparison of spectrums, it can be known that among the 13 characteristic absorption peaks of the traditional Chinese medicine formula composition for improving depression emotion and sleep of the present invention, the peaks having retention times of 26.717 min and 45.426 min are the corresponding active ingredients, which are hyperoside and paeonol, respectively.

Example 6 Determination of the Fingerprint Spectrum of Hypericum perforatum Extract

[0089] 1. Reagents: acetonitrile (HPLC, Wokai); methanol (HPLC, Wokai); phosphoric acid was analytically pure; and UPLC purified water was self-made by Milli-Q Pure Water System.

[0090] 2. Instrument: Agilent Infinity 1260 (CA, USA).

[0091] 3. Preparation of a sample of Hypericum perforatum extract: 0.71 g of Hypericum perforatum extract was accurately weighed and placed into a 100 mL triangular conical flask, and 25 mL of 50% methanol was added, weighed, ultrasonically extracted for 20 min, and then re-weighed. 50% methanol was added to make up the lost weight, filtered, and then a certain volume of filtrate was taken and passed through a 0.45 m microfiltration membrane to obtain the sample solution of Hypericum perforatum extract.

[0092] 4. Chromatographic conditions: the chromatographic conditions and determination methods are the same as those in “(1) Determination of a reference fingerprint spectrum of the traditional Chinese medicine formula composition for improving depression emotion and sleep” in Example 5.

[0093] 5. Determination: The above sample solution of Hypericum perforatum extract was taken and injected into the liquid chromatograph, and the fingerprint spectrum of Hypericum perforatum extract was obtained according to the high performance liquid chromatography method. There are 9 absorption peaks of the main chemical ingredients, which are:

[0094] Peak 1 having an average retention time RT of 11.591 min, an RSD of 0.95%, and an average peak area of 1303.790, which accounts for 3.863% of the peak area.

[0095] Peak 2 having an average retention time RT of 12.299 min, an RSD of 0.86%, and an average peak area of 2686.452, which accounts for 9.402% of the peak area;

[0096] Peak 3 having an average retention time RT of 13.189 min, an RSD of 1.12%, and an average peak area of 895.530, which accounts for 3.134% of the peak area;

[0097] Peak 4 having an average retention time RT of 18.793 min, an RSD of 1.03%, and an average peak area of 538.669, which accounts for 1.885% of the peak area;

[0098] Peak 5 having an average retention time RT of 25.879 min, an RSD of 0.87%, and an average peak area of 4029.912, which accounts for 14.104% of the peak area;

[0099] Peak 6 having an average retention time RT of 26.838 min, an RSD of 0.92%, and an average peak area of 3122.853, which accounts for 10.930% of the peak area;

[0100] Peak 7 having an average retention time RT of 27.434 min, an RSD of 0.87%, and an average peak area of 3520.778, which accounts for 12.322% of the peak area;

[0101] Peak 8 having an average retention time RT of 30.911 min, an RSD of 1.01%, and an average peak area of 1604.837, which accounts for 5.617% of the peak area; and

[0102] Peak 9 having an average retention time RT of 41.111 min, an RSD of 0.78%, and an average peak area of 1040.855, which accounts for 3.643% of the peak area.

Example 7 Determination of the Fingerprint Spectrum of Acanthopanax senticosus

[0103] 1. The reagents and instruments in this Example were the same as those in Example 5.

[0104] 2. Preparation of a sample of Acanthopanax senticosus extract: 0.23 g of Acanthopanax senticosus extract was accurately weighed and placed into a 100 mL triangular conical flask, and 25 mL of 50% methanol was added, weighed, ultrasonically extracted for 20 min, and then re-weighed. 50% methanol was added to make up the lost weight, filtered, and then a certain volume of filtrate was taken and passed through a 0.45 m microfiltration membrane to obtain the sample solution of Acanthopanax senticosus extract.

[0105] 3. Chromatographic conditions: the chromatographic conditions and determination methods were the same as those in “(1) Determination of a reference fingerprint spectrum of the traditional Chinese medicine formula composition for improving depression emotion and sleep” in Example 5.

[0106] 4. Determination: The above sample solution of Acanthopanax senticosus extract was taken and injected into the liquid chromatograph, and the fingerprint spectrum of Acanthopanax senticosus extract was obtained according to the high performance liquid chromatography method. There are 7 absorption peaks of the main chemical components, which are:

[0107] Peak 1 having an average retention time RT of 3.869 min, an RSD of 1.12%, and an average peak area of 583.333, which accounts for 4.466% of the peak area;

[0108] Peak 2 having an average retention time RT of 6.993 min, an RSD of 0.95%, and an average peak area of 272.368, which accounts for 2.085% of the peak area;

[0109] Peak 3 having an average retention time RT of 7.777 min, an RSD of 0.85%, and an average peak area of 296.854, which accounts for 2.273% of the peak area;

[0110] Peak 4 having an average retention time RT of 9.625 min, an RSD of 0.96%, and an average peak area of 361.169, which accounts for 2.765% of the peak area;

[0111] Peak 5 having an average retention time RT of 10.930 min, an RSD of 0.88%, and an average peak area of 3627.005, which accounts for 27.767% of the peak area;

[0112] Peak 6 having an average retention time RT of 12.300 min, an RSD of 0.85%, and an average peak area of 726.493, which accounts for 5.562% of the peak area; and

[0113] Peak 7 having an average retention time RT of 13.186 min, an RSD of 0.90%, and an average peak area of 750.645, which accounts for 5.747% of the peak area.

Example 8 Methodological Verification of Quality Control of the Traditional Chinese Medicine Formula Composition for Improving Depression Emotion and Sleep

1. Preparation of Reference Solution

[0114] An appropriate amount of hyperoside was taken and dissolved in methanol to prepare a reference solution with a concentration of 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL, 0.0625 mg/mL, and 0.03125 mg/mL.

[0115] An appropriate amount of paeonol reference substance was taken and dissolved in methanol to prepare a reference solution with a concentration of 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL, 0.0625 mg/mL, and 0.03125 mg/mL.

2. Production of Standard Curve and Determination of Linear Range

[0116] An appropriate amount of hyperoside standard solution was taken and filtered through a 0.45 μm microfiltration membrane. 10 μL was injected, respectively, based on the set chromatographic conditions. The linear regression equation and linear range of hyperoside were obtained by drawing the standard curve with the chromatographic peak area as the ordinate and the concentration as the abscissa.

[0117] The regression equation of hyperoside is Y=10335.76985X-77.2047, R.sup.2=0.99985; and the regression equation of paeonol is: y=46735.024x-302.7421 (R.sup.2=0.99993). There is a good linear relationship between the concentration and the peak area of hyperoside and paeonol in the linear range.

3. Precision Experiment

[0118] The above hyperoside standard solution and paeonol standard solution were precisely taken and determined by HPLC, with consecutive injected 5 times for 10 μL each time, and determined for the peak area RSD value to test the precision of the high performance liquid chromatograph. For hyperoside, the RSD of the retention time is 0.06% and the RSD of the peak area is 1.07%; and for paeonol, the RSD of the retention time is 0.03% and the RSD of the peak area is 0.23%.

4. Stability Experiment

[0119] Samples of the traditional Chinese medicine composition were taken and determined by HPLC at 0 h, 3 h, 6 h, 9 h, 12 h, and 15 h respectively according to the above chromatographic conditions, with an injection volume of 10 μL. The peak area was integrated to calculate the relative standard deviation of the integrated value and test the stability. The RSD of the peak area of hyperoside was 0.35% and the RSD of the peak area of paeonol was 0.08%.

5. Reproducibility Experiment

[0120] The sample from the same batch was taken to prepare a tested solution according to the preparation method of the tested solution, and determined by HPLC according to the above chromatographic conditions, with an injection volume of 10 μL. The peak area was integrated to calculate the relative standard deviation and test the reproducibility. For hyperoside, the RSD of the retention time is 0.08% and the RSD of the peak area is 0.28%; and for paeonol, the RSD of the retention time is 0.06% and the RSD of the peak area is 0.11%.

6. Sample Recovery Experiment

[0121] 5 samples of the traditional Chinese medicine composition with known content were weighed, and a certain amount of hyperoside and paeonol reference substances was added respectively. Then 5 samples were prepared according to the preparation method of the tested solution, and each sample was continuously injected with an injection volume of 10 μL for each injection. The content of hyperoside and paeonol was determined by the standard curve method, and the average recovery rate of the reference substance was calculated, respectively. The RSD of hyperoside content is 0.52%; and the RSD of paeonol content is 0.19%.

Comparative Example 1

[0122] Compared with Example 5, other conditions remain unchanged, except for mobile phase and elution gradient:

[0123] Mobile phase: 0.1% formic acid aqueous solution (A)-acetonitrile (B); and

[0124] Elution gradient: 0-10 min, 5%-10% B; 10-20 min, 10%-20% B; 20-30 min, 20%-35% B; 30-40 min, 35%-55% B; 40-45 min, 55%-95% B; 45-50 min, 95% B; 50-55 min, 95%-5% B; and 55-65 min, 5% B.

[0125] However, the resolution of the chromatographic peak obtained under this condition was low, and the quality control of the traditional Chinese medicine composition could not be achieved.

Comparative Example 2

[0126] Compared with Example 5, other conditions remain unchanged, except for the gradient conditions:

[0127] 0.1% phosphoric acid aqueous solution (A)-acetonitrile (B); and

[0128] 0-20 min, 10%-20% B; 20-30 min, 20%-25% B; 30-40 min, 25%-40% B; 40-50 min, 40%-10% B; and 50-60 min, 10% B.

[0129] However, the resolution of the chromatographic peak obtained under this condition was low, and the quality control of the traditional Chinese medicine composition could not be achieved.

Comparative Example 3

[0130] Compared with Example 5, other conditions remain unchanged, except for the column temperature and elution conditions:

[0131] Column temperature: 25° C.;

[0132] Mobile phase: 0.1% phosphoric acid aqueous solution (A)-acetonitrile (B); and

[0133] Gradient: 0-20 min, 10%-20% B; 20-30 min, 20%-25% B; 30-40 min, 25%-40% B; 40-50 min, 40%-10% B; and 50-60 min, 10% B.

[0134] However, the resolution of the chromatographic peak obtained under this condition was low, and the quality control of the traditional Chinese medicine composition could not be achieved.

Comparative Example 4

[0135] Compared with Example 5, other conditions remain unchanged, except for flow rate, mobile phase and elution gradient:

[0136] Flow rate: 0.8 ml/min,

[0137] Mobile phase: 0.1% formic acid aqueous solution (A)-acetonitrile (B); and

[0138] Elution gradient: 0-10 min, 5%-10% B; 10-20 min, 10%-20% B; 20-30 min, 20%-35% B; 30-40 min, 35%-55% B; 40-45 min, 55%-95% B; 45-50 min, 95% B; 50-55 min, 95%-5% B; and 55-65 min, 5% B.

[0139] However, the resolution of the chromatographic peak obtained under this condition was low, and the quality control of the traditional Chinese medicine composition could not be achieved.

Comparative Example 5

[0140] Compared with Example 5, other conditions remain unchanged, except for mobile phase and elution gradient:

[0141] Mobile phase: water (A)-acetonitrile (B); and

[0142] Elution gradient: 0-10 min, 10%-14% B; 10-20 min, 14%-20% B; 20-30 min, 20%-25% B; 30-40 min, 25%-40% B; 40-42 min, 40%-60% B; 42-50 min, 60% B; 50-52 min, 60%-10% B; and 52-60 min, 10% B.

[0143] However, the resolution of the chromatographic peak obtained under this condition was low, and the quality control of the traditional Chinese medicine composition could not be achieved.

[0144] In addition, various other separation conditions were also tried in the present invention and the obtained chromatographic peak resolutions were low, and the quality control of the traditional Chinese medicine composition could not be achieved.

[0145] The foregoing description is merely preferred embodiments of the present invention and it should be noted that several improvements and modifications can be made by those ordinary skilled in the art without departing from the principles of the present invention, and these improvements and modifications should also be deemed to be within the protection scope of the present invention.