Method for product quality control and fingerprint detection of epimedium brevicornu complex

Abstract

The present invention discloses a method for product quality control and fingerprint detection of an epimedium brevicornu complex. The method uses high performance liquid chromatography, and can effectively realize the quality control of products containing traditional Chinese medicine components, and especially stable control of the quality of products containing a large quantity of non-traditional Chinese medicine components in formulas. Through step-by-step quality control, product quality fluctuation is reduced and stable quality is ensured. Meanwhile, the method is simple and convenient, does not need additional instruments and standards, saves the cost and is more conducive to actual production.

Claims

1. A method for product quality control and fingerprint detection of an epimedium brevicornu complex, the method for product quality control and fingerprint detection of raw materials of the complex comprising: Step 1: a. preparation of a standard solution by placing 10 mg of an icariin standard into a 10 ml volumetric flask, dissolving in methanol, and making constant volume; b. preparation of an extract sample solution by placing 0.1 g of epimedium brevicornu extract into a conical flask with a stopper; adding 50 ml of methanol; weighing the materials; conducting ultrasonic treatment for 30 min; weighing the materials again; making up the lost weight with the methanol; shaking up; filtering; standing; c. providing content determination under chromatographic condition 1: wherein mobile phase A is acetonitrile, mobile phase B is water; wherein detection wavelength is 270 nm; column temperature is 30° C.; flow rate is 1 ml/min; sample injection volume is 10 μl; and sample injection is performed twice; wherein isocratic elution conditions are: TABLE-US-00015 mobile phase A mobile phase B time, min %, acetonitrile %, water 0-15 30 70; d. making a standard curve by diluting the standard solution by ½ gradient to obtain 5 concentration gradients, analyzing according to the chromatographic conditions, and drawing the standard curve; taking the extract sample solution, analyzing according to the chromatographic condition 1 and calculating the content of the icariin through the standard curve; e) providing fingerprint conditions of the epimedium brevicomu extract: (1) accurately weighing 0.1 g of epimedium brevicornu extract into 50 ml volumetric flask; dissolving in methanol; conducting ultrasonic treatment for 30 min; placing at room temperature; making constant volume; filtering; concentrating; and passing through a membrane; (2) utilizing the following chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler; the flow rate is 1 ml/min; the wavelength is 269 nm; the column temperature is 30° C.; the sample injection volume is 10 μl; and the sample injection is performed once; TABLE-US-00016 time, min 0-17 17-35 35-65 65-70 70-85 acetonitrile, % 15-23 23 23-50 50-15 15; a. further preparation of the standard solution: taking 6 mg of tanshinone IIA standard into 10 ml volumetric flask, dissolving in methanol, and making constant volume; b. preparation of the extract sample solution: taking 0.1 g of salviae miltiorrhizae extract; putting the extract into a conical flask with a stopper; adding 50 ml of methanol; weighing the materials; conducting ultrasonic treatment for 30 min; weighing the materials again; making up the lost weight with the methanol; shaking up; filtering; standing; c. providing content determination under chromatographic condition 2: wherein mobile phase A is acetonitrile, mobile phase B is water; detection wavelength is 270 nm; column temperature is 20° C.; flow rate is 1 ml/min; sample injection is 10 μl; and sample injection is performed twice; wherein the gradient elution conditions are: TABLE-US-00017 mobile phase A mobile phase B time, min %, acetonitrile %, water 0-6 61 39   6-20 61-90 39-10   20-20.5 90-61 10-39 20.5-30   61 39; d. making a standard curve: diluting the standard solution by ½ gradient to obtain 5 concentration gradients, analyzing according to the chromatographic conditions, and drawing the standard curve; taking the extract sample solution, analyzing according to the chromatographic conditions, and calculating the content of the tanshinone IIA through the standard curve; providing fingerprint conditions of the salviae miltiorrhizae extract: (1) accurately weighing 0.1 g of salviae miltiorrhizae extract into 50 ml volumetric flask; dissolving in methanol; conducting ultrasonic treatment for 30 min; placing at room temperature; making constant volume; filtering; concentrating; and passing through a membrane; (2) chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler; the flow rate is 1 ml/min; the wavelength is 270 nm; the column temperature is room temperature; the sample injection volume is 10 μl; and the sample injection is performed once; TABLE-US-00018 time, min 0-20 20-50 50-52 52-62 acetonitrile, % 20-60 60-80 80-20 20; Step 3: a. preparation of the standard solution by taking 8 mg of a protodioscin standard into 10 ml volumetric flask, dissolving in methanol, and making constant volume; b. preparation of the extract sample solution: taking 0.1 g of dioscorea nipponica makino extract; putting the extract into a conical flask with a stopper; adding 50 ml of methanol; weighing the materials; conducting ultrasonic treatment for 30 min; weighing the materials again; making up the lost weight with the methanol; shaking up; filtering; standing; and taking the supernatant; c. providing content determination under chromatographic condition 3: wherein octadecylsilane chemically bonded silica is used as a filler; wherein mobile phase A is acetonitrile, mobile phase B is water; and isocratic elution is performed; wherein detection wavelength is 203 nm; column temperature is 30° C.; flow rate is 1 ml/min; sample injection volume is 10 μl; and sample injection is performed twice; isocratic elution conditions used under chromatographic condition 3: TABLE-US-00019 mobile phase A mobile phase B time, min %, acetonitrile %, water 0-10 37 63 taking the standard solution and the extract sample solution, analyzing according to the chromatographic conditions, and calculating the content of the protodioscin through a single-point method; further providing fingerprint conditions of dioscorea nipponica makino extract: (1) accurately weight 0.1 g of dioscorea nipponica makino extract into 50 ml volumetric flask; dissolving in methanol; conducting ultrasonic treatment for 30 min; placing at room temperature; making constant volume; filtering; concentrating; and passing through a membrane; (2) chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler; flow rate is 1 ml/min; wavelength is 210 nm; column temperature is 30° C.; sample injection volume is 10 μl; and sample injection is performed once; TABLE-US-00020 time, min 0-10 10-20 20-40 40-55 55-65 65-70 70-80 acetonitrile, % 20-25 25-35 35-80 80-100 100 100-20 20.

2. The method for product quality control and fingerprint detection of the epimedium brevicornu complex according to claim 1, wherein the epimedium brevicornu complex is composed of the following raw materials in parts by weight: 1.2 parts of glucosamine, 0.4 part of collagen, 0.2 part of chondroitin sulfate and 0.35 part of traditional Chinese medicine extract; the traditional Chinese medicine extract comprises the following components in parts by weight: 7-12 parts of epimedium brevicornu, 0.5-3 parts of salviae miltiorrhizae, 6.5-9 parts of Dioscorea nipponica makino, 1-5 parts of rhizoma drynariae and 1-5 parts of dodder.

3. A method for product quality control and fingerprint detection of an epimedium brevicornu complex, the method for product quality control and fingerprint detection of the epimedium brevicornu complex comprising: Step 1: providing fingerprint detection under chromatographic conditions 1: a chromatographic column adopts octadecylsilane chemically bonded silica as a filler; mobile phase A is acetonitrile, mobile phase B is water; detection wavelength is 210-270 nm; column temperature is 30° C.; flow rate is 1 ml/min; sample injection volume is 10 μl; and gradient elution conditions: TABLE-US-00021 mobile phase A mobile phase B time, min %, acetonitrile %, water 0-5  5-20 95-80  5-15 20-30 80-70 15-35 30-50 70-50 35-40 50-60 50-40 40-50 60-80 40-20 50-60  80-100 20-0  providing product content detection under chromatographic condition 2: mobile phase A is acetonitrile, mobile phase B is water, acetonitrile: water=30:70, and gradient elution; detection wavelength is 270 nm; column temperature is 30° C.; flow rate is 1 ml/min; sample injection volume is 10 μl; and sample injection is performed twice; Step 2: preparation of a test article: accurately weighing 1 g of epimedium brevicornu complex of claim 2; putting the composition into a conical flask with a stopper; adding 500 ml of methanol; weighing the materials; conducting ultrasonic treatment for 30 min; weighing the materials again; making up the lost weight with the methanol; shaking up; filtering; concentrating the filtrate to 50 ml; standing; Step 3: preparation of the standard solution: taking 10 mg of icariin standard into 10 ml volumetric flask, dissolving in methanol, and making constant volume; making a standard curve: diluting the standard solution by ½ gradient to obtain 5 concentration gradients: 0.0625 mg/ml, 0.125 mg/ml, 0.25 mg/ml, 0.5 mg/ml and 1.0 mg/ml; analyzing according to the chromatographic condition 2; and drawing the standard curve; taking the supernatant obtained in step 2, analyzing according to the chromatographic condition 2, and calculating the content through the standard curve; taking the supernatant obtained in step 2, and analyzing according to the chromatographic condition 1 to obtain a fingerprint.

4. The method for product quality control and fingerprint detection of the epimedium brevicornu complex according to claim 3, wherein the fingerprint of the epimedium brevicornu complex is: the fingerprint comprises 15 common peaks, and the relative retention times of the peaks are: (1) 3.263, (2) 16.857, (3) 17.785, (4) 20.299, (5) 23.178, (6) 23.747, (7) 24.167, (8S) 25.357, (9) 27.064, (10) 28.092, (11) 37.908, (12) 41.202, (13) 53.441, (14) 54.928 and (15) 58.238.

5. The method for product quality control and fingerprint detection of the epimedium brevicornu complex according to claim 1, wherein the methanol is a 100% methanol solution.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) To more clearly describe the technical solution in the embodiments of the present invention or in the prior art, the drawings required to be used in the description of the embodiments or the prior art will be simply presented below. Apparently, the drawings in the following description are merely the embodiments of the present invention, and for those ordinary skilled in the art, other drawings can also be obtained according to the provided drawings without contributing creative labor.

(2) FIG. 1 is a standard curve chart of content determination of icariin for raw material detection in embodiment 1;

(3) FIG. 2 is a standard curve chart of content determination of tanshinone IIA for raw material detection in embodiment 1; and

(4) FIG. 3 is a standard curve chart of content determination of icariin for complex detection in embodiment 1.

DETAILED DESCRIPTION

(5) The technical solution in embodiments of the present invention will be clearly and fully described below. Apparently, the described embodiments are merely part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those ordinary skilled in the art without contributing creative labor will belong to the protection scope of the present invention.

Embodiment 1

(6) Embodiment 1 of the present invention discloses a method for product quality control and fingerprint detection of an epimedium brevicornu complex. The adopted technical solution is as follows:

(7) The epimedium brevicornu complex is composed of the following raw materials: 1200 g of glucosamine, 400 g of collagen, 200 g of chondroitin sulfate and 350 g of traditional Chinese medicine extract. The traditional Chinese medicine extract comprises the following components: 153 g of epimedium brevicornu, 11 g of salviae miltiorrhizae, 153 g of Dioscorea nipponica makino, 16 g of rhizoma drynariae and 17 g of dodder.

(8) (1) Product quality control and fingerprint detection of the traditional Chinese medicine extract:

(9) S1, icariin:

(10) a. preparation of a standard solution: taking 10 mg of icariin standard into 10 ml volumetric flask, dissolving in methanol, and making constant volume;

(11) b. preparation of an extract sample solution: taking 0.1 g of epimedium brevicornu extract; putting the extract into a conical flask with a stopper, adding 50 ml of methanol; weighing the materials; conducting ultrasonic treatment for 30 min; weighing the materials again; making up the lost weight with the methanol; shaking up; filtering, standing; and taking the supernatant;

(12) c. content determination:

(13) chromatographic conditions:

(14) mobile phase A is acetonitrile, mobile phase B is water, acetonitrile:water=30:70, and isocratic elution;

(15) detection wavelength is 270 nm; column temperature is 30° C.; flow rate is 1 ml/min; sample injection volume is 10 μl; and sample injection is performed twice;

(16) isocratic elution conditions:

(17) TABLE-US-00008 mobile phase A mobile phase B time, min %, acetonitrile %, water 0-15 30 70;

(18) d. making of a standard curve: diluting the standard solution by ½ gradient to obtain 5 concentration gradients which are respectively 0.0625 mg/ml, 0.125 mg/ml, 0.25 mg/ml, 0.5 mg/ml and 1.0 mg/ml, analyzing according to the chromatographic conditions, and drawing the standard curve; taking the extract sample solution, analyzing according to the chromatographic conditions, and calculating the content of the icariin through the standard curve, wherein the content of the icariin is calculated as 20.5% according to the sample peak area and the drawn standard curve.

(19) Fingerprint conditions of the epimedium brevicornu extract:

(20) (1) sample preparation: accurately weighing 0.1 g of epimedium brevicornu extract into 50 ml volumetric flask; dissolving in methanol; conducting ultrasonic treatment for 30 min; placing at room temperature; making constant volume; filtering; concentrating; and passing through a membrane.

(21) (2) Chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler; the flow rate is 1 ml/min; the wavelength is 269 nm; the column temperature is 30° C.; the sample injection volume is 10/1; and the sample injection is performed once;

(22) TABLE-US-00009 time/min 0-17 17-35 35-65 65-70 70-85 acetonitrile (%) 15-23 23 23-50 50-15 15

(23) The similarity calculation software is a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine specified by the Chinese Pharmacopoeia Commission. After determination, the similarity with the standard fingerprint is 0.99996.

(24) S2, Tanshinone IIA:

(25) a. preparation of the standard solution: taking 6 mg of tanshinone IIA standard into 10 ml volumetric flask, dissolving in methanol, and making constant volume;

(26) b. preparation of the extract sample solution: taking 0.1 g of salviae miltiorrhizae extract; putting the extract into a conical flask with a stopper, adding 50 ml of methanol; weighing the materials; conducting ultrasonic treatment for 30 min; weighing the materials again; making up the lost weight with the methanol; shaking up; filtering; standing; and taking the supernatant;

(27) c. content determination:

(28) chromatographic conditions:

(29) mobile phase A is acetonitrile, mobile phase B is water, and gradient elution;

(30) detection wavelength is 270 nm; column temperature is 20° C.; flow rate is 1 ml/min; sample injection volume is 10 μl; and sample injection is performed twice;

(31) gradient elution conditions:

(32) TABLE-US-00010 mobile phase A mobile phase B time, min %, acetonitrile %, water 0-6 61 39   6-20 61-90 39-10   20-20.5 90-61 10-39 20.5-30   61 39;

(33) d. making of a standard curve: diluting the standard solution by ½ gradient to obtain 5 concentration gradients which are respectively 0.0375 mg/ml, 0.075 mg/ml, 0.15 mg/ml, 0.3 mg/ml and 0.6 mg/ml, analyzing according to the chromatographic conditions, and drawing the standard curve;

(34) taking the extract sample solution, analyzing according to the chromatographic conditions, and calculating the content of the tanshinone IIA through the standard curve, wherein the content of the tanshinone IIA is calculated as 5.05% according to the sample peak area and the drawn standard curve.

(35) Fingerprint conditions of the salviae miltiorrhizae extract:

(36) (1) chromatographic conditions: accurately weighing 0.1 g of salviae miltiorrhizae extract into 50 ml volumetric flask; dissolving in methanol; conducting ultrasonic treatment for 30 min; placing at room temperature; making constant volume; filtering, concentrating, and passing through a membrane.

(37) (2) Chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler; the flow rate is 1 ml/min; the wavelength is 270 nm; the column temperature is room temperature; the sample injection volume is 10 μl; and the sample injection is performed once.

(38) TABLE-US-00011 time/min 0-20 20-50 50-52 52-62 acetonitrile (%) 20-60 60-80 80-20 20

(39) The similarity calculation software is a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine specified by the Chinese Pharmacopoeia Commission. After determination, the similarity with the standard fingerprint is 0.998.

(40) S3, Protodioscin:

(41) a. preparation of the standard solution: taking 8 mg of protodioscin standard into 10 ml volumetric flask, dissolving in methanol, and making constant volume;

(42) b. preparation of the extract sample solution: taking 0.1 g of Dioscorea nipponica makino extract; putting the extract into a conical flask with a stopper, adding 50 ml of methanol; weighing the materials; conducting ultrasonic treatment for 30 min; weighing the materials again; making up the lost weight with the methanol; shaking up; filtering, standing; and taking the supernatant;

(43) c. content determination:

(44) chromatographic conditions:

(45) octadecylsilane chemically bonded silica is used as a filler;

(46) mobile phase A is acetonitrile, mobile phase B is water, acetonitrile:water=37:63, and isocratic elution;

(47) detection wavelength is 203 nm; column temperature is 30° C.; flow rate is 1 ml/min; sample injection volume is 10 μl; and sample injection is performed twice;

(48) isocratic elution conditions:

(49) TABLE-US-00012 mobile phase A mobile phase B time, min %, acetonitrile %, water 0-10 37 63

(50) taking the standard solution and the extract sample solution, analyzing according to the chromatographic conditions, and calculating the content of the protodioscin through a single-point method, wherein the content of the protodioscin is calculated as 40.22% according to the single-point method.

(51) Fingerprint conditions of Dioscorea nipponica makino extract:

(52) (1) chromatographic conditions: accurately weighing 0.1 g of Dioscorea nipponica makino extract into 50 ml volumetric flask; dissolving in methanol; conducting ultrasonic treatment for 30 min; placing at room temperature; making constant volume; filtering, concentrating, and passing through a membrane;

(53) (2) chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler, flow rate is 1 ml/min; wavelength is 210 nm; column temperature is 30° C.; sample injection volume is 10 μl; and sample injection is performed once;

(54) TABLE-US-00013 time/min 0-10 10-20 20-40 40-55 55-65 65-70 70-80 acetonitrile 20-25 25-35 35-80 80-100 100 100-20 20 (%)

(55) The similarity calculation software is a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine specified by the Chinese Pharmacopoeia Commission. After determination, the similarity with the standard fingerprint is 0.9993.

(56) (2) Product quality control and fingerprint detection of the epimedium brevicornu complex:

(57) S1, fingerprint detection of chromatographic condition 1:

(58) a chromatographic column adopts octadecylsilane chemically bonded silica as a filler;

(59) mobile phase A is acetonitrile, mobile phase B is water, and gradient elution;

(60) detection wavelength is 210-270 nm; column temperature is 30° C.; flow rate is 1 ml/min; and sample injection volume is 101 μl;

(61) gradient elution conditions:

(62) TABLE-US-00014 mobile phase A mobile phase B time, min %, acetonitrile %, water 0-5  5-20 95-80  5-15 20-30 80-70 15-35 30-50 70-50 35-40 50-60 50-40 40-50 60-80 40-20 50-60  80-100 20-0; 

(63) product content detection of chromatographic condition 2:

(64) mobile phase A is acetonitrile, mobile phase B is water, acetonitrile:water=30:70, and gradient elution;

(65) detection wavelength is 270 nm; column temperature is 30° C.; flow rate is 1 ml/min; sample injection volume is 10 μl; and sample injection is performed twice;

(66) S2, preparation of a test article

(67) accurately weighing 1 g of epimedium brevicornu complex; putting the composition into a conical flask with a stopper, adding 500 ml of methanol; weighing the materials; conducting ultrasonic treatment for 30 min; weighing the materials again; making up the lost weight with the methanol; shaking up; filtering; concentrating the filtrate to 50 ml; standing; and taking the supernatant;

(68) S3: preparation of the standard solution

(69) taking 10 mg of icariin standard into 10 ml volumetric flask, dissolving in methanol, and making constant volume;

(70) making of a standard curve: diluting the standard solution by ½ gradient to obtain 5 concentration gradients: 0.0625 mg/ml, 0.125 mg/ml, 0.25 mg/ml, 0.5 mg/ml and 1.0 mg/ml; analyzing according to the chromatographic condition 2; and drawing the standard curve;

(71) taking the supernatant obtained in step S2, analyzing according to the chromatographic condition 2, and calculating the content through the standard curve;

(72) taking the supernatant obtained in step S2, and analyzing according to the chromatographic condition 1 to obtain a fingerprint.

(73) The content of the icariin is calculated as 1.61% according to the sample peak area and the drawn standard curve.

(74) Fingerprint detection of the epimedium brevicornu complex:

(75) the fingerprint comprises 15 common peaks, and the relative retention times of the peaks are:

(76) (1) 3.263, (2) 16.857, (3) 17.785, (4) 20.299, (5) 23.178, (6) 23.747, (7) 24.167, (8S) 25.357, (9) 27.064, (10) 28.092, (11) 37.908, (12) 41.202, (13) 53.441, (14) 54.928 and (15) 58.238.

(77) The similarity calculation software is a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine specified by the Chinese Pharmacopoeia Commission. After determination, the similarity with the standard fingerprint is 0.994.

Embodiment 2

(78) Embodiment 2 of the present invention discloses a method for product quality control and fingerprint detection of an epimedium brevicornu complex. The adopted technical solution is as follows:

(79) The epimedium brevicornu complex is composed of the following raw materials: 1200 g of glucosamine, 400 g of collagen, 200 g of chondroitin sulfate and 350 g of traditional Chinese medicine extract. The traditional Chinese medicine extract comprises the following components: 165 g of epimedium brevicornu, 10 g of salviae miltiorrhizae, 144 g of Dioscorea nipponica makino, 16 g of rhizoma drynariae and 15 g of dodder.

(80) (1) Product quality control and fingerprint detection of the traditional Chinese medicine extract:

(81) S1, icariin:

(82) a. preparation of a standard solution;

(83) b. preparation of an extract sample solution;

(84) c. content determination;

(85) making of a standard curve: diluting the standard solution by ½ gradient to obtain 5 concentration gradients which are respectively 0.075 mg/ml, 0.15 mg/ml, 0.3 mg/ml, 0.6 mg/ml and 1.2 mg/ml, analyzing according to the chromatographic conditions, and drawing the standard curve;

(86) calculating the content of the icariin as 20.5% according to the sample peak area and the drawn standard curve.

(87) Fingerprint of the epimedium brevicornu extract:

(88) The similarity calculation software is a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine specified by the Chinese Pharmacopoeia Commission. After determination, the similarity with the standard fingerprint is 0.9998.

(89) S2, Tanshinone IIA:

(90) taking the extract sample solution, analyzing according to the chromatographic conditions, and calculating the content of the tanshinone IIA through the standard curve. The content of the tanshinone IIA is calculated as 5.06% according to the sample peak area and the drawn standard curve.

(91) Fingerprint of the salviae miltiorrhizae extract:

(92) The similarity calculation software is a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine specified by the Chinese Pharmacopoeia Commission. After determination, the similarity with the standard fingerprint is 0.9991.

(93) S3, Protodioscin:

(94) taking the standard solution and the extract sample solution, analyzing according to the chromatographic conditions, and calculating the content of the protodioscin through a single-point method. The content of the protodioscin is calculated as 40.24% according to the single-point method.

(95) Fingerprint of the Dioscorea nipponica makino extract:

(96) The similarity calculation software is a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine specified by the Chinese Pharmacopoeia Commission. After determination, the similarity with the standard fingerprint is 0.9995.

(97) (2) Product quality control and fingerprint detection of the epimedium brevicornu complex:

(98) The content of the icariin is calculated as 1.73% according to the sample peak area and the drawn standard curve.

(99) Fingerprint detection of the epimedium brevicornu complex:

(100) the fingerprint comprises 15 common peaks, and the relative retention times of the peaks are:

(101) (1) 3.271, (2) 16.850, (3) 17.792, (4) 20.323, (5) 23.181, (6) 23.762, (7) 24.190, (8S) 25.362, (9) 27.077, (10) 28.012, (11) 37.934, (12) 41.216, (13) 53.443, (14) 54.939 and (15) 58.244.

(102) The similarity calculation software is a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine specified by the Chinese Pharmacopoeia Commission. After determination, the similarity with the standard fingerprint is 0.99992.

Embodiment 3

(103) Embodiment 3 of the present invention discloses a method for product quality control and fingerprint detection of an epimedium brevicornu complex. The adopted technical solution is as follows:

(104) The epimedium brevicornu complex is composed of the following raw materials: 2400 g of glucosamine, 800 g of collagen, 400 g of chondroitin sulfate and 700 g of traditional Chinese medicine extract. The traditional Chinese medicine extract comprises the following components: 245 g of epimedium brevicornu, 35 g of salviae miltiorrhizae, 315 g of Dioscorea nipponica makino, 35 g of rhizoma drynariae and 70 g of dodder.

(105) (1) Product quality control and fingerprint detection of the traditional Chinese medicine extract:

(106) S1, icariin:

(107) a. preparation of a standard solution;

(108) b. preparation of an extract sample solution;

(109) c. content determination;

(110) making of a standard curve: diluting the standard solution by ½ gradient to obtain 5 concentration gradients which are respectively 0.075 mg/ml, 0.15 mg/ml, 0.3 mg/ml, 0.6 mg/ml and 1.2 mg/ml, analyzing according to the chromatographic conditions, and drawing the standard curve;

(111) calculating the content of the icariin as 20.3% according to the sample peak area and the drawn standard curve.

(112) Fingerprint of the epimedium brevicornu extract:

(113) The similarity calculation software is a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine specified by the Chinese Pharmacopoeia Commission. After determination, the similarity with the standard fingerprint is 0.99999.

(114) S2, Tanshinone IIA:

(115) taking the extract sample solution, analyzing according to the chromatographic conditions, and calculating the content of the tanshinone IIA through the standard curve. The content of the tanshinone IIA is calculated as 5.07% according to the sample peak area and the drawn standard curve.

(116) Fingerprint of the salviae miltiorrhizae extract:

(117) The similarity calculation software is a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine specified by the Chinese Pharmacopoeia Commission. After determination, the similarity with the standard fingerprint is 0.992.

(118) S3, Protodioscin:

(119) taking the standard solution and the extract sample solution, analyzing according to the chromatographic conditions, and calculating the content of the protodioscin through a single-point method. The content of the protodioscin is calculated as 40.30% according to the single-point method.

(120) Fingerprint of the Dioscorea nipponica makino extract:

(121) The similarity calculation software is a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine specified by the Chinese Pharmacopoeia Commission. After determination, the similarity with the standard fingerprint is 0.9989.

(122) (2) Product quality control and fingerprint detection of the epimedium brevicornu complex:

(123) The content of the icariin is calculated as 1.29% according to the sample peak area and the drawn standard curve.

(124) Fingerprint detection of the epimedium brevicornu complex:

(125) the fingerprint comprises 15 common peaks, and the relative retention times of the peaks are:

(126) (1) 3.263, (2) 16.846, (3) 17.782, (4) 20.301, (5) 23.174, (6) 23.752, (7) 24.183, (8S) 25.359, (9) 27.068, (10) 28.012, (11) 37.911, (12) 41.206, (13) 53.444, (14) 54.935 and (15) 58.241.

(127) The similarity calculation software is a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine specified by the Chinese Pharmacopoeia Commission. After determination, the similarity with the standard fingerprint is 0.9991.

(128) Each embodiment in the description is described in a progressive way. The difference of each embodiment from each other is the focus of explanation. The same and similar parts among all of the embodiments can be referred to each other.

(129) The above description of the disclosed embodiments enables those skilled in the art to realize or use the present invention. Many modifications to these embodiments will be apparent to those skilled in the art. The general principle defined herein can be realized in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principle and novel features disclosed herein.