Determination of 8 chemical components in the Miao medicine Panax ginseng

Abstract

This invention discloses a method for determining the content of eight chemical components in the Miao medicine *Gynostemma pentaphyllum*. The method utilizes a UPLC (upper-lowering chromatography) approach to simultaneously determine the content of these eight chemical components in *Gynostemma pentaphyllum*. The method can detect the content of proanthocyanidins B1, B, catechins, B4, B2, epicatechin, cinnamyl tannin B1, and A2. This analytical method exhibits high sensitivity, good specificity, and is stable and feasible. It also reveals that the accumulation of chemical components in *Gynostemma pentaphyllum* varies under different growth environments.

Description

Technical Field

[0001] This invention relates to methods for determining multiple components of medicinal materials, particularly methods for determining the content of eight chemical components in Miao medicine blood ginseng. 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] Extensive research has been conducted on the components of blood ginseng, but there are no reports on the simultaneous detection of the contents of eight chemical components in blood ginseng: proanthocyanidin B1, proanthocyanidin B, catechin, proanthocyanidin B4, proanthocyanidin B2, epicatechin, cinnamyl tannin B1, and proanthocyanidin A2. Methodological verification shows that this analytical method has high sensitivity and is stable and feasible. Summary of the Invention

[0004] The purpose of this invention is to provide a method for determining the content of eight chemical components in the Miao medicine blood ginseng. This method can detect the content of these eight chemical components: 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 determining the content of eight chemical components in Miao medicine blood ginseng, 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) The test solution and the mixed reference solution ② were subjected to UPLC determination under chromatographic conditions to effectively separate proanthocyanidins B1, proanthocyanidins B, catechins, proanthocyanidins B4, proanthocyanidins B2, epicatechins, cinnamic tannins B1, and proanthocyanidins A2 from ginseng, and the contents of proanthocyanidins B1, proanthocyanidins B, catechins, proanthocyanidins B4, proanthocyanidins B2, epicatechins, cinnamic tannins B1, and proanthocyanidins A2 were determined.

[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: 0-120 min, 7%-7% B; 120-130 min, 7%-11% B; 130-140 min, 11%-11% B; 140-150 min, 11%-12% B; 150-160 min, 12%-12% B; 160-165 min, 12%-15% B.

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

[0019] This invention establishes a UPLC method for the simultaneous determination of eight chemical components in blood ginseng, a traditional Chinese medicine. The method can detect the content of these eight chemical components: 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.

[0020] This invention can provide a scientific basis for the quality evaluation of blood ginseng based on its antioxidant effects. The content determination results show that the contents of eight chemical components in 41 batches of blood ginseng medicinal materials vary greatly. Among them, the contents of proanthocyanidin B4, proanthocyanidin B2, epicatechin, cinnamic tannin B1, and proanthocyanidin A2 are relatively high, while the contents of proanthocyanidin B1, proanthocyanidin B3, and catechin are relatively low. This result indicates that there are certain differences in the accumulation of chemical components in blood ginseng medicinal materials under different growth environments. Attached Figure Description

[0021] Figure 1 UPLC chromatogram of mixed reference standards (1 is proanthocyanidin B1; 2 is proanthocyanidin B3; 3 is catechin; 4 is proanthocyanidin B4; 5 is proanthocyanidin B2; 6 is epicatechin; 7 is cinnamyl tannin B1; 8 is proanthocyanidin A2);

[0022] Figure 2 UPLC chromatogram of the test solution (1 is proanthocyanidin B1; 2 is proanthocyanidin B3; 3 is catechin; 4 is proanthocyanidin B4; 5 is proanthocyanidin B2; 6 is epicatechin; 7 is cinnamyl tannin B1; 8 is proanthocyanidin A2);

[0023] Figure 3: Chromatogram of blank reagent for specificity test;

[0024] Figure 4 Linearity was examined using the standard curve of proanthocyanidin B1;

[0025] Figure 5 Linearity was examined using the standard curve of proanthocyanidin B3;

[0026] Figure 6 Linearity analysis was conducted using the catechin standard curve.

[0027] Figure 7 Linearity was examined using the standard curve of proanthocyanidin B4;

[0028] Figure 8 Linearity was examined using the standard curve of proanthocyanidin B2;

[0029] Figure 9 Linearity was examined using the standard curve of epicatechin;

[0030] Figure 10 Linearity was investigated using the standard curve of cinnamon tannin B1;

[0031] Figure 11 Linearity was investigated using the standard curve of proanthocyanidin A2. Detailed Implementation

[0032] 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.

[0033] Example 1:

[0034] (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;

[0035] (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;

[0036] (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;

[0037] (4) Chromatographic conditions: InfinityLab Poroshell 120Aq-C was used. 18 The chromatographic column (2.1×150 mm, 2.7 μm) 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: 0–120 min, 7%–7% B; 120–130 min, 7%–11% B; 130–140 min, 11%–11% B; 140–150 min, 11%–12% B; 150–160 min, 12%–12% B; 160–165 min, 12%–15% B; the flow rate was 0.2 mL / min, the detection wavelength was 202 nm, the column temperature was 30 °C, and the injection volume was 3 μL.

[0038] (5) Take the test solution and the mixed reference solution ② and perform UPLC determination according to the chromatographic conditions respectively, and calculate the contents of proanthocyanidin B1, proanthocyanidin B, catechin, proanthocyanidin B4, proanthocyanidin B2, epicatechin, cinnamic tannin B1 and proanthocyanidin A2.

[0039] Example 2:

[0040] (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;

[0041] (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.

[0042] (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;

[0043] (4) Chromatographic conditions: InfinityLab Poroshell 120Aq-C was used. 18 The chromatographic column (2.1×150 mm, 2.7 μm) 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: 0–120 min, 7%–7% B; 120–130 min, 7%–11% B; 130–140 min, 11%–11% B; 140–150 min, 11%–12% B; 150–160 min, 12%–12% B; 160–165 min, 12%–15% B; the flow rate was 0.2 mL / min, the detection wavelength was 202 nm, the column temperature was 30 °C, and the injection volume was 3 μL.

[0044] (5) Take the test solution and the mixed reference solution ② and perform UPLC determination according to the chromatographic conditions respectively, and calculate the contents of proanthocyanidin B1, proanthocyanidin B, catechin, proanthocyanidin B4, proanthocyanidin B2, epicatechin, cinnamic tannin B1 and proanthocyanidin A2.

[0045] Example 3:

[0046] (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;

[0047] (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.

[0048] (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;

[0049] (4) Chromatographic conditions: InfinityLab Poroshell 120Aq-C was used. 18The chromatographic column (2.1×150 mm, 2.7 μm) 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: 0–120 min, 7%–7% B; 120–130 min, 7%–11% B; 130–140 min, 11%–11% B; 140–150 min, 11%–12% B; 150–160 min, 12%–12% B; 160–165 min, 12%–15% B; the flow rate was 0.2 mL / min, the detection wavelength was 202 nm, the column temperature was 30 °C, and the injection volume was 3 μL.

[0050] (5) Take the test solution and the mixed reference solution ② and perform UPLC determination according to the chromatographic conditions respectively, and calculate the contents of proanthocyanidin B1, proanthocyanidin B, catechin, proanthocyanidin B4, proanthocyanidin B2, epicatechin, cinnamic tannin B1 and proanthocyanidin A2.

[0051] Example 4:

[0052] (1) Preparation of mixed reference solution ①: Accurately weigh 7.12 mg, 11.89 mg and 18.99 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 1.424 mg / mL proanthocyanidin B1, 2.378 mg / mL proanthocyanidin B and 3.798 mg / mL catechin respectively;

[0053] (2) Preparation of mixed reference solution ②: Accurately weigh 10.05 mg, 6.12 mg, 5.02 mg, 16.08 mg, and 8.95 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 a solution of mass concentration. A mixed reference solution containing 0.01424 mg / mL proanthocyanidin B1, 0.02378 mg / mL proanthocyanidin B, 0.03798 mg / mL catechin, 0.01005 mg / mL proanthocyanidin B4, 0.0612 mg / mL proanthocyanidin B2, 0.0502 mg / mL epicatechin, 0.1608 mg / mL cinnamyl tannin B1, and 0.0895 mg / mL proanthocyanidin A2, respectively.

[0054] (3) Preparation of test solution: Take blood ginseng sample, crush it, pass it through No. 2 sieve, weigh 0.4998g 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;

[0055] (4) Chromatographic conditions: InfinityLab Poroshell 120Aq-C was used. 18 The chromatographic column (2.1×150 mm, 2.7 μm) 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: 0–120 min, 7%–7% B; 120–130 min, 7%–11% B; 130–140 min, 11%–11% B; 140–150 min, 11%–12% B; 150–160 min, 12%–12% B; 160–165 min, 12%–15% B; the flow rate was 0.2 mL / min, the detection wavelength was 202 nm, the column temperature was 30 °C, and the injection volume was 3 μL.

[0056] (5) Take the test solution and the mixed reference solution ② and perform UPLC determination according to the chromatographic conditions respectively, and calculate the contents of proanthocyanidin B1, proanthocyanidin B, catechin, proanthocyanidin B4, proanthocyanidin B2, epicatechin, cinnamic tannin B1 and proanthocyanidin A2.

[0057] The inventor conducted numerous experiments; the following are some of the experimental studies.

[0058] 1. Instruments and Reagents

[0059] 1.1 Instruments

[0060] 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.).

[0061] 1.2 Reagents

[0062] 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.

[0063] 2 Test Samples

[0064] 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.

[0065] Table 1 Sample Source Information

[0066]

[0067]

[0068] 3. Methods and Results

[0069] 3.1 Preparation of mixed reference solution

[0070] Accurately weigh 7.07 mg, 12.03 mg, and 19.01 mg of proanthocyanidin B1, proanthocyanidin B3, and catechin reference standards, respectively, and place them in a 5 mL volumetric flask. Dilute to the mark with 50% methanol solution, shake well, and prepare a mixed reference solution① with mass concentrations of 1.414 mg / mL, 2.404 mg / mL, and 3.802 mg / mL, respectively. Accurately weigh 10.01 mg, 6.03 mg, and 5.0 mg of proanthocyanidin B4, proanthocyanidin B2, epicatechin, cinnamyl tannin B1, and proanthocyanidin A2 reference standards, respectively. 2 mg, 16.00 mg, and 9.05 mg were placed in a 100 mL volumetric flask and labeled ②. Then, 1 mL of solution ① was accurately pipetted into ②, and 50% methanol was added to the mark. The mixture was shaken well to prepare a mixed reference solution ② with mass concentrations of 0.0141 mg / mL, 0.0240 mg / mL, 0.0380 mg / mL, 0.1001 mg / mL, 0.0603 mg / mL, 0.0502 mg / mL, 0.1600 mg / mL, and 0.0905 mg / mL, respectively. This solution was then prepared for use. 3.2 Preparation of the test solution

[0071] Accurately weigh approximately 0.5 g of ginseng sample powder (passed through a No. 2 sieve) and place it in a 50 mL stoppered conical flask. Add 10 mL of 60% methanol solution and extract by sonication for 40 min (power 400 W, frequency 40 kHz). Cool the sample, replenish the weight loss with 60% methanol solution, shake well, filter through a 0.22 μm microporous membrane, and collect the filtrate to obtain the test solution.

[0072] 3.3 Chromatographic conditions

[0073] Using InfinityLab Poroshell 120Aq-C 18 The chromatographic column (2.1 × 150 mm, 2.7 μm) used 0.1% phosphoric acid aqueous solution (A)-acetonitrile (B) as the mobile phase, and gradient elution was performed. The elution program was: 0–120 min, 7%–7% B; 120–130 min, 7%–11% B; 130–140 min, 11%–11% B; 140–150 min, 11%–12% B; 150–160 min, 12%–12% B; 160–165 min, 12%–15% B; the flow rate was 0.2 mL / min, the detection wavelength was 202 nm, the column temperature was 30 ℃, and the injection volume was 3 μL. The chromatograms of the mixed reference solution and the test solution are shown below. Figure 1 , Figure 2 .

[0074] 3.4 Methodological Examination

[0075] 3.4.1 Specificity Test

[0076] To further examine the rationality of the experiment, a specificity test was conducted. The results are shown below. Figure 3 The results showed that the blank solution had no chromatographic peaks at the corresponding retention times of the reference solution at a detection wavelength of 202 nm, indicating no interference and good specificity.

[0077] 3.4.2 Examination of Linear Relationships

[0078] Accurately weigh 5.02 mg and 5.01 mg of proanthocyanidin B4 and cinnamyl tannin B1, respectively, and place them in a 5 mL volumetric flask. Dilute to the mark with 50% methanol, shake well, and prepare a mixed reference solution ③ with mass concentrations of 1.0040 mg / mL and 1.0020 mg / mL, respectively. Accurately pipette 1 mL of mixed reference solution ① from section “3.1”, place it in a 10 mL volumetric flask, dilute to the mark with 50% methanol, shake well, and prepare a mixed reference solution ④. Accurately pipette 1 mL of mixed reference solution ② from section “3.1”, place it in a 500 mL volumetric flask, dilute to the mark with 50% methanol, shake well, and prepare a mixed reference solution ⑤ for later use. Before injection, filter all mixed reference solutions through a 0.22 μm microporous membrane and inject according to the chromatographic conditions in section “3.3”. Mixed reference solution ② was injected at concentrations of 0.5 μL, 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 10 μL, and 12 μL; mixed reference solution ③ was injected at concentrations of 0.2 μL, 0.4 μL, 0.6 μL, 0.9 μL, and 1 μL; mixed reference solution ④ was injected at concentrations of 1 μL and 1.5 μL; and mixed reference solution ⑤ was injected at concentration of 1 μL. Peak areas were measured and recorded. Linear regression was performed with the analyte injection amount (μg) as the x-axis (X) and peak area as the y-axis (Y). A standard curve was plotted, and the regression equation was obtained. The correlation coefficients r were all greater than 0.9999, indicating a good linear relationship. The results are shown in Table 2. The limit of detection (LOD) was defined as the injection amount with a signal-to-noise ratio (S / N) of approximately 3 in the chromatogram, and the limit of quantitation (LOQ) was defined as the injection amount with a signal-to-noise ratio (S / N) of approximately 10. The results are shown in Table 2, and the standard curve is shown in Table 2. Figures 4-11 .

[0079] Table 2. Experimental Study on Linear Relationship

[0080]

[0081] 3.4.3 Precision Test

[0082] Take a sample of mixed reference standard ② and inject it six times consecutively under the chromatographic conditions described in section “3.3”, recording the peak areas. Calculate the peak areas of proanthocyanidins B1, B3, catechins, B4, B2, epicatechin, cinnamyl tannin B1, and A2. The RSD values ​​are 0.05%, 0.25%, 0.59%, 0.18%, 0.08%, 0.04%, 0.12%, and 0.47%, respectively, indicating good instrument precision.

[0083] 3.4.4 Stability Test

[0084] The same batch of sample powder S41 was prepared according to the method in section "3.2". Chromatographic measurements were performed under the conditions in section "3.3" at 0 h, 5 h, 10 h, 15 h, 20 h, 25 h, 36 h, and 50 h, and the peak areas were recorded. The peak areas of proanthocyanidins B1, B3, catechins, B4, B2, epicatechin, cinnamyl tannin B1, and A2 in the test solution were calculated. The RSD values ​​were 0.70%, 1.18%, 0.66%, 0.95%, 0.72%, 1.14%, 0.86%, and 0.58%, respectively, indicating that the test solution was stable within 50 h.

[0085] 3.4.5 Repeatability Test

[0086] Take 6 portions of the same batch of sample powder S41, prepare them according to the method in section "3.2", and analyze them under the chromatographic conditions in section "3.3". Record the peak area. The mass fractions of proanthocyanidins B1, B3, catechins, B4, B2, epicatechin, cinnamyl tannin B1, and A2 in the test solution were calculated. Their average values ​​were 0.1482 mg / g, 0.4681 mg / g, 0.3605 mg / g, 2.0701 mg / g, 0.7648 mg / g, 0.7217 mg / g, 3.3803 mg / g, and 2.6447 mg / g, respectively, with RSD values ​​of 2.63%, 2.26%, 1.89%, 2.04%, 2.98%, 2.05%, 2.45%, and 2.31%, respectively. This indicates that the method has good repeatability in determining the content of the above eight components in ginseng.

[0087] 3.4.6 Spiking Recovery Test

[0088] Accurately weigh 7.40 mg of proanthocyanidin B1, 5.85 mg of proanthocyanidin B3, 9.01 mg of catechin, 5.29 mg of proanthocyanidin B4, 9.56 mg of proanthocyanidin B2, 9.02 mg of epicatechin, 8.45 mg of cinnamyl tannin B1, and 6.61 mg of proanthocyanidin A2 into volumetric flasks of 200 mL, 50 mL, 100 mL, 10 mL, 50 mL, 50 mL, 10 mL, and 10 mL respectively. Dilute to volume with 50% methanol solution and shake well to obtain eight single reference solutions. The mass concentrations are 0.0370 mg / mL, 0.1170 mg / mL, 0.0901 mg / mL, 0.5290 mg / mL, 0.1912 mg / mL, 0.1804 mg / mL, 0.8450 mg / mL, and 0.6610 mg / mL, respectively. Take 0.25g of the same batch of sample powder S1, accurately weigh it, and place it in a 50mL stoppered conical flask. Make a total of 6 portions. Accurately pipette 1mL of each of the above 8 reference solutions, then accurately add 2mL of 60% methanol solution, weigh it, prepare it according to the method under "3.2", inject it for analysis according to the chromatographic conditions under "3.3", and record the peak area. The recoveries of proanthocyanidins B1, B3, catechins, B4, B2, epicatechin, cinnamic tannin B1, and A2 were 104.08%, 104.05%, 98.90%, 102.90%, 96.91%, 104.33%, 98.39%, and 99.91%, respectively, with RSDs of 0.68%, 0.90%, 0.77%, 1.56%, 1.41%, 0.53%, 2.35%, and 1.66%, respectively. These results indicate that the method has good accuracy. The results are shown in Table 3.

[0089] Table 3 Results of the sample recovery test

[0090]

[0091]

[0092]

[0093] 4. Sample Determination

[0094] The content of the samples was determined according to the methods in sections “3.2” and “3.3”, and the results are shown in Table 4.

[0095] Table 4. Content of 8 chemical components in 41 batches of blood ginseng medicinal materials

[0096]

[0097]

[0098] 5. Conclusions and Discussion

[0099] 5.1 Conclusion

[0100] This invention establishes a method for simultaneously determining the content of eight chemical components in 41 batches of ginseng by UPLC, which can provide a scientific basis for the quality evaluation of ginseng based on its antioxidant efficacy. The content determination results show that the content of the eight chemical components in the 41 batches of ginseng varies greatly. Among them, the contents of proanthocyanidins B4, proanthocyanidins B2, epicatechin, cinnamyl tannin B1, and proanthocyanidins A2 are relatively high, while the contents of proanthocyanidins B1, proanthocyanidins B3, and catechins are relatively low. This result indicates that there are certain differences in the accumulation of chemical components in ginseng under different growth environments.

[0101] 5.2 Discussion

[0102] 5.2.1 Selection of detection wavelength

[0103] A PDA detector was used to perform full-wavelength scanning of single reference solutions for the content determination of eight chemical components. It was found that the maximum absorption wavelength of proanthocyanidin B1, proanthocyanidin B3, catechin, proanthocyanidin B4, proanthocyanidin B2, epicatechin, cinnamyl tannin B1, and proanthocyanidin A2 was 202 nm. Therefore, the content of the eight chemical components was simultaneously detected at a wavelength of 202 nm.

[0104] 5.2.2 Selection of Mobile Phase

[0105] The mobile phases of 0.1% phosphoric acid aqueous solution (A)-acetonitrile (B), 0.1% formic acid aqueous solution (A)-acetonitrile (B), 0.1% acetic acid aqueous solution (A)-acetonitrile (B), 0.1% phosphoric acid aqueous solution (A)-methanol (B), and 0.1% formic acid aqueous solution (A)-methanol (B) were investigated. The results showed that the mobile phase of 0.1% phosphoric acid aqueous solution (A)-acetonitrile (B) produced the best peak shape and the best separation effect.

[0106] 5.2.3 Selection of Column Temperature

[0107] The resolution of chromatograms was investigated at column temperatures of 25℃, 30℃, and 35℃. The results showed that the peak resolution was best at 30℃. Therefore, a column temperature of 30℃ was selected.

[0108] 5.2.4 Selection of Injection Volume

[0109] The results showed that the chromatographic peak response was low when the injection volume was 2 μL, and peak tailing occurred when the injection volume was 3.5 μL. The peak shape was optimal when the injection volume was 3 μL. Therefore, an injection volume of 3 μL was selected.

[0110] 5.2.5 Selection of Flow Rate

[0111] The resolution of chromatographic peaks was investigated at flow rates of 0.15 mL / min, 0.2 mL / min, and 0.25 mL / min. The results showed that a flow rate of 0.2 mL / min resulted in good separation and was also economical in terms of reagents. Therefore, a flow rate of 0.2 mL / min was selected.

Claims

1. A method for determining the content of eight chemical components in Miao medicine blood ginseng, characterized in that: The method is performed according to 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 label it as ②. Then accurately pipette 1 mL of mixed reference solution ① into 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 at a flow rate of 0.2 mL / min and a detection wavelength of 202 nm. The column temperature was 30 °C and the injection volume was 3 µL. The elution program was as follows: 0–120 min, 7%–7% B; 120–130 min, 7%–11% B; 130–140 min, 11%–11% B; 140–150 min, 11%–12% B; 150–160 min, 12%–12% B; 160–165 min, 12%–15% B. (5) The test solution and the mixed reference solution ② were subjected to UPLC determination under chromatographic conditions to effectively separate proanthocyanidins B1, proanthocyanidins B, catechins, proanthocyanidins B4, proanthocyanidins B2, epicatechins, cinnamic tannins B1, and proanthocyanidins A2 from ginseng, and the contents of proanthocyanidins B1, proanthocyanidins B, catechins, proanthocyanidins B4, proanthocyanidins B2, epicatechins, cinnamic tannins B1, and proanthocyanidins A2 were determined.

2. The method for determining the content of eight chemical components in Miao medicine blood ginseng 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 determining the content of eight chemical components in Miao medicine 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 determining the content of eight chemical components in Miao medicine blood ginseng as described in claim 1, characterized in that: In step (2), 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, cinnamic tannin B1, and proanthocyanidin A2 reference standards, respectively, and place them in a 100 mL volumetric flask, labeled ②. Then, accurately pipette 1 mL of mixed reference solution ① into ②, add 45-55% methanol to the mark, shake well, and 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 determining the content of eight chemical components in Miao medicine 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, cinnamic tannin B1, and proanthocyanidin A2 reference standards, respectively, and place them in a 100 mL volumetric flask, labeled ②. Then, accurately pipette 1 mL of the mixed reference solution ① into ②, add 50% methanol to the mark, and shake well to prepare a solution with mass concentrations of 0.0141 mg / mL for proanthocyanidin B1, 0.0240 mg / mL for proanthocyanidin A2, and 0.0240 mg / mL for proanthocyanidin B2. 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 determining the content of eight chemical components in Miao medicine 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 determining the content of eight chemical components in Miao medicine 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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