A method for establishing an HPLC fingerprint of blood ginseng, a traditional Chinese medicine.

Abstract

This invention discloses a method for establishing an HPLC fingerprint of Miao medicine ginseng. The method involves UPLC fingerprint analysis and similarity analysis of 41 batches of ginseng samples, revealing 36 common peaks. Eight of these common peaks were identified using a reference standard: peak 2 (proanthocyanidin B1), peak 4 (proanthocyanidin B3), peak 6 (catechin), peak 8 (proanthocyanidin B4), peak 9 (proanthocyanidin B2), peak 11 (epicatechin), peak 14 (cinnamyl tannin B1), and peak 29 (proanthocyanidin A2). The similarity between the fingerprints of 39 batches of ginseng samples and the reference standard was greater than 0.900, indicating that the quality of ginseng from different origins is relatively stable.

Description

Technical Field

[0001] This invention relates to methods for determining multiple components of medicinal materials, and in particular to a method for establishing an HPLC fingerprint of blood ginseng, a Miao medicine. Background Technology

[0002] Blood ginseng (Indigofera stachyodes, ISs) is the dried root of Indigofera atachyoides Lindl, a plant in the genus Indigofera of the legume family. It is a commonly used folk medicine of the Miao ethnic group and was included in the "Quality Standards of Traditional Chinese Medicine and Ethnic Medicines of Guizhou Province" in 2003. It has a long history of use. It tastes sweet and slightly bitter, is warm in nature, and enters the liver, kidney, and large intestine meridians. It has the effects of nourishing yin and replenishing deficiency, regulating menstruation and stopping bleeding, and promoting blood circulation and relaxing muscles. It is mainly used to treat metrorrhagia and menorrhagia. It is mainly distributed in Yunnan, Guizhou, Fujian, Guangxi and other places, especially in Guiyang, Anshun, southwestern Guizhou and southeastern Guizhou.

[0003] Currently, fingerprinting is one of the most effective methods for evaluating traditional Chinese medicine (TCM). However, there are few studies on UPLC fingerprinting or multiple-evaluation methods for blood ginseng. Therefore, ultra-high performance liquid chromatography (UHPLC) was chosen for this study to fill this gap and to shorten injection time and improve detection efficiency. This study enriches the information on common components of blood ginseng and, through fingerprint similarity software analysis, indicates that there are certain differences in the components contained in blood ginseng from different regions, which may be related to factors such as origin and growth years. Summary of the Invention

[0004] The purpose of this invention is to provide a method for establishing an HPLC fingerprint of blood ginseng, a traditional Chinese medicine, which can detect the content of eight chemical components in blood ginseng: proanthocyanidin B1, proanthocyanidin B, catechin, proanthocyanidin B4, proanthocyanidin B2, epicatechin, cinnamyl tannin B1, and proanthocyanidin A2. This analytical method has high sensitivity, good specificity, and is stable and feasible.

[0005] The technical solution of this invention: A method for establishing an HPLC fingerprint of blood ginseng from Miao medicine, the method comprising the following steps:

[0006] (1) Preparation of mixed reference solution ①: Accurately weigh proanthocyanidin B1, proanthocyanidin B3 and catechin reference standards, place them in a volumetric flask, add methanol solution to make up to the mark, shake well, and prepare mixed reference solution ①.

[0007] (2) Preparation of mixed reference solution ②: Accurately weigh proanthocyanidin B4, proanthocyanidin B2, epicatechin, cinnamyl tannin B1 and proanthocyanidin A2, place them in a volumetric flask, then accurately pipette 1 mL of mixed reference solution ① into mixed reference solution ②, add methanol to make up to the mark, shake well, and prepare mixed reference solution ②.

[0008] (3) Preparation of test solution: Take blood ginseng sample, crush it, pass it through No. 2 sieve, weigh the powder, accurately weigh it, put it in a stoppered conical flask, add methanol solution, weigh it, extract it by ultrasound, cool it, make up the weight loss with methanol solution, shake it well, filter it with a 0.22μm microporous membrane, and take the filtrate to obtain the test solution.

[0009] (4) Chromatographic conditions: InfinityLab Poroshell 120Aq-C was used. 18 The chromatographic column (2.1×150mm, 2.7μm) was used with 0.1% phosphoric acid aqueous solution as phase A and acetonitrile as phase B, and gradient elution was performed at a flow rate of 0.2mL / min and a detection wavelength of 202nm; the column temperature was 30℃ and the injection volume was 3μL.

[0010] (5) Perform UPLC determination of the test solution and mixed reference solution ② under chromatographic conditions. Import the chromatograms into the 2012 version of the software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" of the National Pharmacopoeia Commission in AIA format. Using the peak area and retention time of the cinnamyl tannin B1 peak as a reference, calculate the RSD values ​​of the relative peak area and relative retention time of each common peak.

[0011] In step (1) above, the preparation of the mixed reference solution ① is as follows: accurately weigh 6.5-7.5 mg, 11.5-12.5 mg, and 18.5-19.5 mg of proanthocyanidin B1, proanthocyanidin B3, and catechin reference standards, place them in a 5 mL volumetric flask, add 45-55% methanol solution to dilute to the mark, shake well, and prepare a mixed reference solution ① with mass concentrations of 1.3-1.5 mg / mL proanthocyanidin B1, 2.3-2.5 mg / mL proanthocyanidin B, and 3.7-3.9 mg / mL catechin, respectively.

[0012] Specifically, in step (1) above, the preparation of the mixed reference solution ① is as follows: accurately weigh 7.07 mg, 12.03 mg, and 19.01 mg of proanthocyanidin B1, proanthocyanidin B3, and catechin reference standards, place them in a 5 mL volumetric flask, add 50% methanol solution to dilute to the mark, shake well, and prepare a mixed reference solution ① with mass concentrations of 1.414 mg / mL proanthocyanidin B1, 2.404 mg / mL proanthocyanidin B, and 3.802 mg / mL catechin, respectively.

[0013] In step (2) above, the preparation of mixed reference solution ② is as follows: Accurately weigh 9.5-10.5 mg, 5.5-6.5 mg, 4.5-5.5 mg, 15.00-17.00 mg, and 8.5-9.5 mg of proanthocyanidin B4, proanthocyanidin B2, epicatechin, cinnamyl tannin B1, and proanthocyanidin A2 reference standards, respectively, and place them in a 100 mL volumetric flask. Then, accurately pipette 1 mL of mixed reference solution ① into mixed reference solution ②, add 45-55% methanol to the mark, shake well, and prepare a solution with a mass concentration of 0.01... A mixed reference solution containing 3-0.015 mg / mL proanthocyanidin B1, 0.0230-0.025 mg / mL proanthocyanidin B, 0.0370-0.0390 mg / mL catechin, 0.095-0.105 mg / mL proanthocyanidin B4, 0.055-0.065 mg / mL proanthocyanidin B2, 0.045-0.055 mg / mL epicatechin, 0.1500-0.1700 mg / mL cinnamyl tannin B1, and 0.085-0.095 mg / mL proanthocyanidin A2.

[0014] Specifically, in step (2) above, the preparation of mixed reference solution ② is as follows: Accurately weigh 10.01 mg, 6.03 mg, 5.02 mg, 16.00 mg, and 9.05 mg of proanthocyanidins B4, B2, epicatechin, cinnamyl tannin B1, and A2 reference standards, respectively, and place them in a 100 mL volumetric flask. Then, accurately pipette 1 mL of mixed reference solution ① into mixed reference solution ②, add 50% methanol to the mark, and shake well. A mixed reference solution was prepared with mass concentrations of 0.0141 mg / mL proanthocyanidin B1, 0.0240 mg / mL proanthocyanidin B, 0.0380 mg / mL catechin, 0.1001 mg / mL proanthocyanidin B4, 0.0603 mg / mL proanthocyanidin B2, 0.0502 mg / mL epicatechin, 0.1600 mg / mL cinnamyl tannin B1, and 0.0905 mg / mL proanthocyanidin A2, respectively.

[0015] In step (3) above, the preparation of the test solution is as follows: Take a blood ginseng sample, crush it, pass it through a No. 2 sieve, weigh 0.49-0.51g of powder, accurately weigh it, place it in a 50mL stoppered conical flask, add 10mL of 55-65% methanol solution, weigh it, extract it by ultrasound for 35-45min, ultrasound power 400W, ultrasound frequency 40KHz, cool it, replenish the weight loss with 55-65% methanol solution, shake it well, filter it through a 0.22μm microporous membrane, and take the filtrate to obtain the test solution.

[0016] Specifically, in step (3) above, the preparation of the test solution is as follows: take a blood ginseng sample, crush it, pass it through a No. 2 sieve, weigh 0.5g of powder, accurately weigh it, place it in a 50mL stoppered conical flask, add 10mL of 60% methanol solution, weigh it, extract it by ultrasound for 40min, ultrasound power 400W, ultrasound frequency 40KHz, cool it, replenish the weight loss with 60% methanol solution, shake it well, filter it through a 0.22μm microporous membrane, and take the filtrate to obtain the test solution.

[0017] In step (4) above, the elution program is as follows: 0-10 min, 9.5%-12% B; 10-40 min, 12%-25% B; 40-41 min, 25%-100% B; 41-42 min, 100%-100% B; flow rate 0.2 mL / min.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] This invention performed UPLC fingerprint analysis and similarity analysis on 41 batches of blood ginseng samples, identifying 36 common peaks. Eight of these common peaks were identified using a reference standard: peak 2 (proanthocyanidin B1), peak 4 (proanthocyanidin B3), peak 6 (catechin), peak 8 (proanthocyanidin B4), peak 9 (proanthocyanidin B2), peak 11 (epicatechin), peak 14 (cinnamyl tannin B1), and peak 29 (proanthocyanidin A2). The similarity between the fingerprints of 39 batches of blood ginseng samples and the reference standard was greater than 0.900, indicating relatively stable quality of blood ginseng from different origins. The similarity of batches S35 and S37 was 0.881 and 0.890, respectively, indicating that these two batches differed from other batches. Attached Figure Description

[0020] Figure 1 : Precision fingerprint overlay image;

[0021] Figure 2 Stability fingerprint overlay;

[0022] Figure 3 : Overlay of repeatable fingerprint patterns;

[0023] Figure 4 UPLC fingerprint overlay images of 41 batches of medicinal materials;

[0024] Figure 5 : 41 batches of blood ginseng medicinal materials comparison fingerprint spectrum (R) (2-proanthocyanidin B1; 4-proanthocyanidin B3; 6-catechin; 8-proanthocyanidin B4; 9-proanthocyanidin B2; 11-epicatechin; 14-cinnamyl tannin B1; 29-proanthocyanidin A2). Detailed Implementation

[0025] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0026] Example 1:

[0027] (1) Preparation of mixed reference solution ①: Accurately weigh 7.07 mg, 12.03 mg and 19.01 mg of proanthocyanidin B1, proanthocyanidin B3 and catechin reference standards respectively, place them in a 5 mL volumetric flask, add 50% methanol solution to make up to the mark, shake well, and prepare a mixed reference solution ① with mass concentrations of 1.414 mg / mL proanthocyanidin B1, 2.404 mg / mL proanthocyanidin B and 3.802 mg / mL catechin respectively;

[0028] (2) Preparation of mixed reference solution ②: Accurately weigh 10.01 mg, 6.03 mg, 5.02 mg, 16.00 mg, and 9.05 mg of proanthocyanidin B4, proanthocyanidin B2, epicatechin, cinnamyl tannin B1, and proanthocyanidin A2 reference standards, respectively, and place them in a 100 mL volumetric flask. Then, accurately pipette 1 mL of mixed reference solution ① into mixed reference solution ②, add 50% methanol to the mark, shake well, and prepare the solution by weight. A mixed reference solution with concentrations of 0.0141 mg / mL proanthocyanidin B1, 0.0240 mg / mL proanthocyanidin B, 0.0380 mg / mL catechin, 0.1001 mg / mL proanthocyanidin B4, 0.0603 mg / mL proanthocyanidin B2, 0.0502 mg / mL epicatechin, 0.1600 mg / mL cinnamyl tannin B1, and 0.0905 mg / mL proanthocyanidin A2, respectively;

[0029] (3) Preparation of test solution: Take blood ginseng sample, crush it, pass it through No. 2 sieve, weigh 0.5g of powder, accurately weigh it, put it in a 50mL stoppered conical flask, add 10mL of 60% methanol solution, weigh it, extract it by ultrasound for 40min, ultrasound power 400W, ultrasound frequency 40KHz, cool it, make up the weight loss with 60% methanol solution, shake it well, filter it through a 0.22μm microporous membrane, and take the filtrate to obtain the test solution;

[0030] (4) Chromatographic conditions: InfinityLab Poroshell 120Aq-C was used. 18The chromatographic column (2.1×150mm, 2.7μm) used a mobile phase of 0.1% phosphoric acid aqueous solution as phase A and acetonitrile as phase B, with gradient elution. The elution program was as follows: 0–10 min, 9.5%–12% B; 10–40 min, 12%–25% B; 40–41 min, 25%–100% B; 41–42 min, 100%–100% B; flow rate 0.2 mL / min; detection wavelength 202 nm; column temperature 30℃; injection volume 3 μL.

[0031] (5)(5) The test solution and the mixed reference solution ② were subjected to UPLC determination under chromatographic conditions. The chromatograms were imported into the 2012 version of the software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" of the National Pharmacopoeia Commission in AIA format. The relative peak area and relative retention time of the cinnamon tannin B1 peak were used as references to calculate the RSD values ​​of the relative peak area and relative retention time of each common peak.

[0032] Example 2:

[0033] (1) Preparation of mixed reference solution ①: Accurately weigh 6.5 mg, 11.5 mg and 18.5 mg of proanthocyanidin B1, proanthocyanidin B3 and catechin reference standards respectively, place them in a 5 mL volumetric flask, add 45% methanol solution to make up to the mark, shake well, and prepare a mixed reference solution ① with mass concentrations of 1.3 mg / mL proanthocyanidin B1, 2.3 mg / mL proanthocyanidin B and 3.7 mg / mL catechin respectively;

[0034] (2) Preparation of mixed reference solution ②: Accurately weigh 9.5 mg, 5.5 mg, 4.5 mg, 15.00 mg, and 8.5 mg of proanthocyanidin B4, proanthocyanidin B2, epicatechin, cinnamon tannin B1, and proanthocyanidin A2 reference standards, respectively, and place them in a 100 mL volumetric flask. Then, accurately pipette 1 mL of mixed reference solution ① into mixed reference solution ②, add 45% methanol to the mark, shake well, and prepare mixed reference solution ② with mass concentrations of 0.013 mg / mL proanthocyanidin B1, 0.0230 mg / mL proanthocyanidin B, 0.0370 mg / mL catechin, 0.095 mg / mL proanthocyanidin B4, 0.055 mg / mL proanthocyanidin B2, 0.045 mg / mL epicatechin, 0.1500 mg / mL cinnamon tannin B1, and 0.085 mg / mL proanthocyanidin A2, respectively.

[0035] (3) Preparation of test solution: Take blood ginseng sample, crush it, pass it through No. 2 sieve, weigh 0.49g of powder, accurately weigh it, put it in a 50mL stoppered conical flask, add 10mL of 55% methanol solution, weigh it, extract it by ultrasound for 35min, ultrasound power 400W, ultrasound frequency 40KHz, cool it, make up the weight loss with 55% methanol solution, shake it well, filter it through a 0.22μm microporous membrane, and take the filtrate to obtain the test solution;

[0036] (4) Chromatographic conditions: InfinityLab Poroshell 120Aq-C was used. 18 The chromatographic column (2.1×150mm, 2.7μm) used a mobile phase of 0.1% phosphoric acid aqueous solution as phase A and acetonitrile as phase B, with gradient elution. The elution program was as follows: 0–10 min, 9.5%–12% B; 10–40 min, 12%–25% B; 40–41 min, 25%–100% B; 41–42 min, 100%–100% B; flow rate 0.2 mL / min; detection wavelength 202 nm; column temperature 30℃; injection volume 3 μL.

[0037] (5) Perform UPLC determination of the test solution and mixed reference solution ② under chromatographic conditions. Import the chromatograms into the 2012 version of the software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" of the National Pharmacopoeia Commission in AIA format. Using the peak area and retention time of the cinnamyl tannin B1 peak as a reference, calculate the RSD values ​​of the relative peak area and relative retention time of each common peak.

[0038] Example 3:

[0039] (1) Preparation of mixed reference solution ①: Accurately weigh 7.5 mg, 12.5 mg and 19.5 mg of proanthocyanidin B1, proanthocyanidin B3 and catechin reference standards respectively, place them in a 5 mL volumetric flask, add 55% methanol solution to make up to the mark, shake well, and prepare a mixed reference solution ① with mass concentrations of 1.5 mg / mL proanthocyanidin B1, 25 mg / mL proanthocyanidin B and 3.9 mg / mL catechin respectively;

[0040] (2) Preparation of mixed reference solution ②: Accurately weigh 10.5 mg, 6.5 mg, 5.5 mg, 17.00 mg, and 9.5 mg of proanthocyanidin B4, proanthocyanidin B2, epicatechin, cinnamon tannin B1, and proanthocyanidin A2 reference standards, respectively, and place them in a 100 mL volumetric flask. Then, accurately pipette 1 mL of mixed reference solution ① into mixed reference solution ②, add 55% methanol to the mark, shake well, and prepare mixed reference solution ② with mass concentrations of 0.015 mg / mL proanthocyanidin B1, 0.025 mg / mL proanthocyanidin B, 0.0390 mg / mL catechin, 0.105 mg / mL proanthocyanidin B4, 0.065 mg / mL proanthocyanidin B2, 0.055 mg / mL epicatechin, 0.1700 mg / mL cinnamon tannin B1, and 0.095 mg / mL proanthocyanidin A2, respectively.

[0041] (3) Preparation of test solution: Take blood ginseng sample, crush it, pass it through No. 2 sieve, weigh 0.51g of powder, accurately weigh it, put it in a 50mL stoppered conical flask, add 10mL of 65% methanol solution, weigh it, extract it by ultrasound for 45min, ultrasound power 400W, ultrasound frequency 40KHz, cool it, make up the weight loss with 65% methanol solution, shake it well, filter it through a 0.22μm microporous membrane, and take the filtrate to obtain the test solution;

[0042] (4) Chromatographic conditions: InfinityLab Poroshell 120Aq-C was used. 18 The chromatographic column (2.1×150mm, 2.7μm) used a mobile phase of 0.1% phosphoric acid aqueous solution as phase A and acetonitrile as phase B, with gradient elution. The elution program was as follows: 0–10 min, 9.5%–12% B; 10–40 min, 12%–25% B; 40–41 min, 25%–100% B; 41–42 min, 100%–100% B; flow rate 0.2 mL / min; detection wavelength 202 nm; column temperature 30℃; injection volume 3 μL.

[0043] (5)(5) The test solution and the mixed reference solution ② were subjected to UPLC determination under chromatographic conditions. The chromatograms were imported into the 2012 version of the software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" of the National Pharmacopoeia Commission in AIA format. The relative peak area and relative retention time of the cinnamon tannin B1 peak were used as references to calculate the RSD values ​​of the relative peak area and relative retention time of each common peak.

[0044] The inventor conducted numerous experiments; the following are some of the experimental studies.

[0045] 1. Instruments and Reagents

[0046] 1.1 Instruments

[0047] Ultra-high performance liquid chromatograph (model: LC-2040C 3D Plus, manufacturer: Shimadzu, Japan), InfinityLabPoroshell 120Aq-C 18 Chromatographic column (2.1×150mm, 2.7μm), FA2204B type 0.001 g electronic balance (Shanghai Tianmei Balance Instrument Co., Ltd.), MS205DU type 0.001 g electronic balance (Mettler Toledo Instruments (China) Co., Ltd.), SB-4200DTD ultrasonic cleaner (Ningbo Xinyi Ultrasonic Equipment Co., Ltd.), SHZ-DIII circulating water vacuum pump (Gongyi Yuhua Instrument Co., Ltd.), RHP-750A multi-functional pulverizer (Zhejiang Ronghao Industry and Trade Co., Ltd.).

[0048] 1.2 Reagents

[0049] Proanthocyanidins B1 (batch number: CYK-Z0382), B2 (batch number: CYK-Z0383), B3 (batch number: CYK-Z0384), B4 (batch number: CYK-Z0385), A2 (batch number: CYK-Z0376), Cinnamyl Tannin B1 (batch number: CYK-Z0392), Catechins (batch number: CYK-Z0875), and Epicatechin (batch number: CYK-Z0876) were all purchased from Wuhan Qiongge Biotechnology Co., Ltd., and the mass fraction of all the above reference standards was greater than 98%. Acetonitrile was chromatographic grade (TEDIA, USA), all other reagents were analytical grade, and purified water was from Guiyang Wahaha Changsheng Beverage Co., Ltd.

[0050] 2 Test Samples

[0051] The blood ginseng samples were collected from Guiyang, Longli, Huishui, and other places in Guizhou Province. Except for S41, which was purchased, all the others were wild-grown. All blood ginseng samples were identified by Professor Wang Bo of Guizhou University of Traditional Chinese Medicine as the dried roots of Indigofera stachyodes Lindl., a plant belonging to the genus Indigofera in the family Leguminosae. Collection information is detailed in Table 1.

[0052] Table 1 Sample Source Information

[0053]

[0054]

[0055]

[0056] 3. Methodological Investigation

[0057] 3.1 Precision Test

[0058] Accurately weigh a sample of ginseng S41 (passed through a No. 2 sieve), prepare the test solution according to the method in Example 1, and inject it continuously 6 times under the chromatographic conditions of Example 1, recording the chromatograms. Import the chromatograms into the National Pharmacopoeia Commission software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version) in AIA format. Using the peak area and retention time of peak 14 as a reference, calculate the RSD values ​​of the relative peak area and relative retention time of each common peak. The results are all less than 3%, indicating good instrument precision. The results are shown in Table 2-5. Figure 1 .

[0059] Table 2 Precision Test - Common Peak Area

[0060]

[0061]

[0062] Table 3 Precision Test - Common Peak Retention Time

[0063]

[0064]

[0065] Table 4 Precision Test - Relative Peak Area of ​​Common Peak

[0066]

[0067]

[0068] Table 5 Precision Test - Relative Retention Time of Common Peak

[0069]

[0070] 3.2 Stability Test

[0071] The same test solution was injected at 0h, 9h, 14h, 20h, 32h, and 48h under the chromatographic conditions of Example 1, and the chromatograms were recorded. The chromatograms were imported into the National Pharmacopoeia Commission software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version) in AIA format, and the RSD values ​​of the relative peak area and relative retention time of each common peak were calculated. The results were all less than 3%, indicating that the test solution was stable within 48h. The results are shown in Tables 6-9. Figure 2 .

[0072] Table 6 Stability Test - Common Peak Area

[0073]

[0074]

[0075] Table 7 Stability Test - Common Peak Retention Time

[0076]

[0077] Table 8 Stability Test - Relative Peak Area of ​​Common Peak

[0078]

[0079]

[0080] Table 9 Stability Test - Relative Retention Time of Common Peak

[0081]

[0082]

[0083] 3.3 Repeatability Test

[0084] Accurately weigh ginseng S41 samples and prepare six test solutions according to the method in Example 1. Inject the solutions sequentially under the chromatographic conditions of Example 1 and record the chromatograms. Import the chromatograms into the National Pharmacopoeia Commission software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version) in AIA format. Calculate the RSD values ​​of the relative peak area and relative retention time of each common peak. The results are all less than 3%, indicating good repeatability of the method. The results are shown in Tables 10-13. Figure 3 .

[0085] Table 10 Repeatability Tests - Common Peak Area

[0086]

[0087]

[0088] Table 11 Repeatability Tests - Common Peak Retention Time

[0089]

[0090]

[0091] Table 12 Repeatability Tests - Relative Peak Areas of Common Peaks

[0092]

[0093]

[0094] Table 13 Repeatability Tests - Relative Retention Times of Common Peaks

[0095]

[0096]

[0097] 4. Experimental Results

[0098] 4.1 Establishment of the fingerprint spectrum of blood ginseng in Miao medicine

[0099] Forty-one batches of ginseng were collected, and test solutions were prepared according to the method in Example 1. Chromatographic analysis was performed under the chromatographic conditions of Example 1, and chromatograms were recorded. Integration was performed using a Shimadzu workstation. The chromatograms of the forty-one batches of ginseng were imported into the National Pharmacopoeia Commission software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version) in AIA format. The chromatogram S1 of the ginseng sample was set as the reference chromatogram. The median method was used for calculation, and after multi-point correction, automatic peak matching was performed to obtain a superimposed fingerprint chromatogram of the forty-one batches of ginseng. A ginseng control fingerprint chromatogram (R) was also generated. (See...) Figure 4-5 Among 41 batches of ginseng samples, 36 common chromatographic peaks of different components were found, numbered 1 to 36 in order of retention time. Through identification with reference standards, 8 chemical components were identified, including peak 2 as proanthocyanidin B1, peak 4 as proanthocyanidin B3, peak 6 as catechin, peak 8 as proanthocyanidin B4, peak 9 as proanthocyanidin B2, peak 11 as epicatechin, peak 14 as cinnamyl tannin B1, and peak 29 as proanthocyanidin A2.

[0100] 4.2 Similarity Analysis

[0101] Similarity analysis was performed on the fingerprint chromatograms of 41 batches of ginseng. Peak 14 (cinnamyl tannin B1) was used as the reference peak, and the retention time and peak area of ​​the reference peak were set to 1. The relative peak area and relative retention time of the common peaks in the 41 batches of ginseng were calculated, and the results are shown in Tables 14-26. The similarity between the 41 batches of ginseng samples and with the control fingerprint chromatogram was calculated using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version), and the results are shown in Tables 27-29.

[0102] Table 14 shows the total peak area of ​​ginseng medicinal materials, batches 1-9.

[0103]

[0104]

[0105] Table 15. Total peak area of ​​41 batches of blood ginseng medicinal materials, Nos. 10-18

[0106]

[0107]

[0108] Table 16. Total peak area of ​​41 batches of blood ginseng medicinal materials, Nos. 19-27

[0109]

[0110]

[0111] Table 17. Total peak area of ​​41 batches of blood ginseng medicinal materials, Nos. 28-36

[0112]

[0113]

[0114] Table 18. Relative peak area of ​​41 batches of blood ginseng medicinal materials, Nos. 1-14

[0115]

[0116]

[0117] Table 19. Relative peak area of ​​41 batches of blood ginseng medicinal materials, Nos. 15-28

[0118]

[0119]

[0120] Table 20: Relative Peak Area of ​​Common Peaks in Batch 41 of Blood Ginseng Medicinal Materials Nos. 29-36

[0121]

[0122]

[0123] Table 21. Peak retention times of 41 batches of blood ginseng medicinal materials, Nos. 1-12

[0124]

[0125]

[0126] Table 22. Peak retention time of 41 batches of blood ginseng medicinal materials, Nos. 13-24.

[0127]

[0128]

[0129] Table 23. Peak retention time of 41 batches of blood ginseng medicinal materials, Nos. 25-36.

[0130]

[0131]

[0132] Table 24. Relative retention times of peaks in 41 batches of ginseng medicinal materials, Nos. 1-12.

[0133]

[0134]

[0135] Table 25. Relative retention times of common peaks in 41 batches of blood ginseng medicinal materials, Nos. 13-24.

[0136]

[0137]

[0138] Table 26. Relative retention times of peaks in batches 41 of ginseng medicinal materials, numbers 25-36.

[0139]

[0140]

[0141] Table 27 Similarity Analysis Results of 41 Batches of Blood Ginseng Medicinal Materials - 1

[0142]

[0143]

[0144] Table 28 Similarity Analysis Results of 41 Batches of Blood Ginseng Medicinal Materials - 2

[0145]

[0146]

[0147] Table 29 Similarity Analysis Results of 41 Batches of Blood Ginseng Medicinal Materials - 3

[0148]

[0149]

[0150] The results showed that the similarity between 39 batches of blood ginseng samples and the control fingerprint spectrum was greater than 0.900, and the similarity between 2 batches of blood ginseng samples was 0.881 and 0.890.

[0151] 5. Conclusions and Discussion

[0152] 5.1 Conclusion

[0153] UPLC fingerprint analysis and similarity analysis were performed on 41 batches of blood ginseng samples, revealing 36 common peaks. Eight of these common peaks were identified using a reference standard: peak 2 (proanthocyanidin B1), peak 4 (proanthocyanidin B3), peak 6 (catechin), peak 8 (proanthocyanidin B4), peak 9 (proanthocyanidin B2), peak 11 (epicatechin), peak 14 (cinnamyl tannin B1), and peak 29 (proanthocyanidin A2). The similarity between the fingerprints of 39 batches of blood ginseng samples and the reference standard was greater than 0.900, indicating relatively stable quality of blood ginseng from different origins. The similarity of batches S35 and S37 was 0.881 and 0.890, respectively, indicating certain differences between these two batches and other batches.

[0154] 5.2 Discussion

[0155] Fingerprint analysis of traditional Chinese medicine (TCM) is one of the most effective methods for evaluating TCM. Currently, there are few studies on UPLC fingerprint analysis or multiple-evaluation methods for blood ginseng. Therefore, ultra-high performance liquid chromatography (UHPLC) was chosen for this study to fill this gap and to shorten the injection time and improve detection efficiency. This study enriched the list of common components in blood ginseng and, through fingerprint similarity software analysis, indicated that there are certain differences in the components contained in blood ginseng from different regions, which may be related to factors such as origin and growth years.

Claims

1. A method for establishing an HPLC fingerprint of ginseng (a traditional Chinese medicine), characterized in that: It includes the following steps: (1) Preparation of mixed reference solution ①: Accurately weigh proanthocyanidin B1, proanthocyanidin B3 and catechin reference standards, place them in a volumetric flask, add methanol solution to make up to the mark, shake well, and prepare mixed reference solution ①. (2) Preparation of mixed reference solution ②: Accurately weigh proanthocyanidin B4, proanthocyanidin B2, epicatechin, cinnamyl tannin B1 and proanthocyanidin A2, place them in a volumetric flask, and then accurately pipette 1 mL of mixed reference solution ① into a volumetric flask, which is marked as ②. Add methanol to make up to the mark, shake well, and prepare mixed reference solution ②. (3) Preparation of test solution: Take blood ginseng sample, crush it, pass it through No. 2 sieve, weigh the powder, accurately weigh it, put it in a stoppered conical flask, add methanol solution, weigh it, extract it by ultrasound, cool it, make up the weight loss with methanol solution, shake it well, filter it with a 0.22 µm microporous membrane, and take the filtrate to obtain the test solution. (4) Chromatographic conditions: InfinityLab Poroshell 120 Aq-C was used. 18 The chromatographic column was 2.1 × 150 mm with a diameter of 2.7 µm. The mobile phase consisted of 0.1% phosphoric acid aqueous solution as phase A and acetonitrile as phase B, with gradient elution. The detection wavelength was 202 nm. The column temperature was 30 °C, and the injection volume was 3 µL. The elution program was as follows: 0–10 min, 9.5%–12% B; 10–40 min, 12%–25% B; 40–41 min, 25%–100% B; 41–42 min, 100%–100% B; flow rate 0.2 mL / min. (5) The test solution and the mixed reference solution ② were subjected to UPLC determination under chromatographic conditions. The chromatograms were imported into the software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" 2012 version of the National Pharmacopoeia Commission software in AIA format. The relative peak area and relative retention time of the cinnamon tannin B1 peak were used as references to calculate the RSD values ​​of the relative peak area and relative retention time of each common peak.

2. The method for establishing an HPLC fingerprint of ginseng (a traditional Chinese medicine) as described in claim 1, characterized in that: In step (1), the preparation of the mixed reference solution ① is as follows: accurately weigh 6.5-7.5 mg, 11.5-12.5 mg, and 18.5-19.5 mg of proanthocyanidin B1, proanthocyanidin B3, and catechin reference standards, place them in a 5 mL volumetric flask, add 45-55% methanol solution to dilute to the mark, shake well, and prepare a mixed reference solution ① with mass concentrations of 1.3-1.5 mg / mL proanthocyanidin B1, 2.3-2.5 mg / mL proanthocyanidin B3, and 3.7-3.9 mg / mL catechin, respectively.

3. The method for establishing an HPLC fingerprint of blood ginseng as described in claim 2, characterized in that: In step (1), the preparation of the mixed reference solution ① is as follows: Accurately weigh 7.07 mg, 12.03 mg, and 19.01 mg of proanthocyanidin B1, proanthocyanidin B3, and catechin reference standards, place them in a 5 mL volumetric flask, add 50% methanol solution to make up to the mark, shake well, and prepare a mixed reference solution ① with mass concentrations of 1.414 mg / mL proanthocyanidin B1, 2.406 mg / mL proanthocyanidin B3, and 3.802 mg / mL catechin, respectively.

4. The method for establishing an HPLC fingerprint of blood ginseng as described in claim 1, characterized in that: In step (2), the preparation of the mixed reference solution ② is as follows: Accurately weigh 9.5-10.5 mg, 5.5-6.5 mg, 4.5-5.5 mg, 15.00-17.00 mg, and 8.5-9.5 mg of proanthocyanidin B4, proanthocyanidin B2, epicatechin, cinnamic tannin B1, and proanthocyanidin A2 reference standards, respectively, and place them in a 100 mL volumetric flask. Then, accurately pipette 1 mL of the mixed reference solution ① into the volumetric flask, label it ②, and add 45-55% methanol to the mark. Shake well to prepare a solution with a mass concentration of 0.013-0.015 mg. A mixed reference solution containing 0.0230-0.025 mg / mL proanthocyanidin B1, 0.0370-0.0390 mg / mL catechin, 0.095-0.105 mg / mL proanthocyanidin B4, 0.055-0.065 mg / mL proanthocyanidin B2, 0.045-0.055 mg / mL epicatechin, 0.1500-0.1700 mg / mL cinnamyl tannin B1, and 0.085-0.095 mg / mL proanthocyanidin A2.

5. The method for establishing an HPLC fingerprint of blood ginseng as described in claim 1, characterized in that: In step (2), the preparation of the mixed reference solution ② is as follows: Accurately weigh 10.01 mg, 6.03 mg, 5.02 mg, 16.00 mg, and 9.05 mg of proanthocyanidin B4, proanthocyanidin B2, epicatechin, cinnamyl tannin B1, and proanthocyanidin A2 reference standards, respectively, and place them in a 100 mL volumetric flask. Then, accurately pipette 1 mL of the mixed reference solution ① into the volumetric flask, label it ②, and add 50% methanol to the mark. Shake well to prepare solutions with mass concentrations of 0.0141 mg / mL for proanthocyanidin B1 and 0.0240 mg / mL for proanthocyanidin A2. A mixed reference solution of 0.0380 mg / mL proanthocyanidin B3, 0.1001 mg / mL proanthocyanidin B4, 0.0603 mg / mL proanthocyanidin B2, 0.0502 mg / mL epicatechin, 0.1600 mg / mL cinnamyl tannin B1, and 0.0905 mg / mL proanthocyanidin A2 ②.

6. The method for establishing an HPLC fingerprint of blood ginseng as described in claim 1, characterized in that: In step (3), the preparation of the test solution is as follows: Take a blood ginseng sample, crush it, pass it through a No. 2 sieve, weigh 0.49-0.51 g of powder, accurately weigh it, place it in a 50 mL stoppered conical flask, add 10 mL of 55-65% methanol solution, weigh it, extract it by ultrasound for 35-45 min, ultrasound power 400 W, ultrasound frequency 40 KHz, cool it, replenish the weight loss with 55-65% methanol solution, shake it well, filter it through a 0.22 µm microporous membrane, and take the filtrate to obtain the test solution.

7. The method for establishing an HPLC fingerprint of blood ginseng as described in claim 6, characterized in that: In step (3), the preparation of the test solution is as follows: Take a blood ginseng sample, crush it, pass it through a No. 2 sieve, weigh 0.5 g of powder, accurately weigh it, place it in a 50 mL stoppered conical flask, add 10 mL of 60% methanol solution, weigh it, extract it by ultrasound for 40 min, ultrasound power 400 W, ultrasound frequency 40 KHz, cool it, replenish the weight loss with 60% methanol solution, shake it well, filter it through a 0.22 µm microporous membrane, and take the filtrate to obtain the test solution.

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