Method for rapid quantitative detection of notoginseng saponins

Abstract

The invention discloses a rapid quantitative detection method for saponins in Panax notoginseng, comprising: extracting a Panax notoginseng sample with methanol to obtain a methanol extract of the Panax notoginseng sample; and analyzing the methanol extract of the Panax notoginseng sample using an ultra-high performance liquid chromatography-diode array detector system; wherein the ultra-high performance liquid chromatography uses an ACQUITY UPLC@BEH-C18 chromatographic column (1.7um×2.1×50mm); the conditions of the ultra-high performance liquid chromatography are as follows: mobile phase A: methanol, mobile phase B: water; gradient elution: 0-5min, 20%A; 5-10min, 20%A-45%A; the acquisition wavelength of the diode array detector is 203nm. The rapid quantitative detection method for saponins in Panax notoginseng of the present invention has accurate and reliable detection results.

Description

Technical Field

[0001] The invention relates to the field of chemical analysis and testing, and in particular to a method for rapid quantitative detection of notoginseng saponins. Background Art

[0002] Panax notoginseng (Burk.) FHChen is a plant of the genus Panax in the Araliaceae family. It is sweet, slightly bitter and warm in nature. It is a precious Chinese medicinal material unique to my country. It has the effects of dispersing blood stasis and stopping bleeding, reducing swelling and relieving pain, stopping bleeding, lowering blood lipids and protecting the liver (China Pharmacopoeia Committee, 2015). In the traditional Chinese medicine industry, Panax notoginseng has become the largest Chinese medicinal material after ginseng. It is the main raw material for Chinese patent medicines such as Yunnan Baiyao, Compound Danshen Drop Pills, and Xuesetong (Li Pinhan, 2016; Li Qian, 2018; Zhao Nana et al., 2018). According to statistics, there are more than 540 Chinese patent medicines with Panax notoginseng as raw materials in China, with more than 3,600 drug batch numbers, involving more than 1,350 pharmaceutical companies (Liu Lihong et al., 2017). Panax notoginseng total saponins (PNSS) play an important role in traditional Chinese medicine and are the main active ingredients of Panax notoginseng. An increasing number of in vivo and in vitro studies have shown that PNSS are widely used to treat various diseases, such as atherosclerosis, acute lung injury, cancer, and cardiovascular disease (Xu et al., 2014; Chen et al., 2016; Fan et al., 2016; Liao et al., 2016; Yang et al., 2016; Huang et al., 2017; Jin et al., 2017; Kitamura et al., 2017; Ratno Budiarto and Chan, 2017; Tian X.-P. et al., 2017). However, the quantitative detection technology of Panax notoginseng quality is relatively backward. The mainstream is still based on the qualitative detection of Panax notoginseng saponin R1, ginsenoside Rg1, Rb1 and Re by thin layer chromatography in the 2015 edition of the Pharmacopoeia, and the quantitative detection of Panax notoginseng saponin R1, ginsenoside Rg1 and Rb1 by liquid chromatography, and the total amount of the three must not be less than 5%. The sample pretreatment method of this method is cumbersome and time-consuming, requiring about 14 hours, and the liquid phase detection time also requires 1 hour, which greatly restricts the detection efficiency of the quality of Panax notoginseng. Patent document CN109061001A discloses a method for detecting ginsenosides in plasma using liquid chromatography-mass spectrometry, which is not suitable for the quality detection of Panax notoginseng. The existing technology detects various saponins in Panax notoginseng by UPLC-MS / MS, but the instrument operation requirements are high, and the cost of the instrument itself is very high, which is not suitable for detection in grassroots market promotion. Summary of the invention

[0003] The present invention aims to establish a rapid quantitative detection method for notoginseng saponins, which is suitable for the detection of diversified notoginseng products, including not only notoginseng powder and notoginseng decoction pieces mainly based on notoginseng roots, but also quality detection of products with notoginseng flowers and notoginseng leaves as raw materials.

[0004] The present invention provides a method for rapid quantitative detection of saponins in Panax notoginseng, comprising:

[0005] Extracting the Panax notoginseng sample with methanol to obtain a methanol extract of the Panax notoginseng sample; and

[0006] The methanol extract of the Panax notoginseng sample is analyzed by using an ultra-high performance liquid chromatography-diode array detector system;

[0007] The conditions of the ultra-high performance liquid chromatography are as follows: the ultra-high performance liquid chromatography uses an ACQUITY UPLC@BEH-C18 chromatographic column (1.7um×2.1×50mm); the conditions of the ultra-high performance liquid chromatography are as follows: mobile phase A: methanol, mobile phase B: water; gradient elution: 0-5min, 20%A; 5-10min, 20%A-45%A; the collection wavelength of the diode array detector is 203nm.

[0008] Wherein, the flow rate of the mobile phase is 0.25 mL / min.

[0009] Wherein, the column temperature of the chromatographic column is 30°C.

[0010] The photoelectric separation of the diode matrix detector is 1.2 nm, and the sampling rate is 10 pt / s.

[0011] Among them, the saponins in the Panax notoginseng are one or more of notoginseng saponin R1, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, ginsenoside Rc, ginsenoside Rb3, ginsenoside Rd, and ginsenoside Rg2.

[0012] Wherein, ultrasonic treatment was performed when the Panax notoginseng sample was extracted with methanol.

[0013] Wherein, jujube saponin B is used as an internal standard in the ultra-high performance liquid chromatography.

[0014] Among them, the ultra-high performance liquid chromatography uses a standard curve of notoginsenoside R1, a standard curve of ginsenoside Rg1, a standard curve of ginsenoside Rb1, a standard curve of ginsenoside Re, a standard curve of ginsenoside Rc, a standard curve of ginsenoside Rb3, a standard curve of ginsenoside Rd and a standard curve of ginsenoside Rg2.

[0015] The rapid quantitative detection method of saponins in Panax notoginseng of the present invention can detect notoginseng saponin R1, ginsenoside Rg1, Rb1 and Re, the four saponins required by the Pharmacopoeia 2015 edition, and can also detect characteristic ginsenosides Rc and Rb3, notoginseng saponin Rd and rare saponin Rg2 in the aerial part of Panax notoginseng, so a total of 8 notoginseng saponins can be detected. The rapid quantitative detection method of saponins in Panax notoginseng of the present invention can simultaneously detect 8 notoginseng saponins in Panax notoginseng samples, and the detection results are accurate and reliable.

[0016] The rapid quantitative detection method of saponins in Panax notoginseng of the present invention utilizes an ultra-high performance liquid chromatography-photodiode array detector (UPLC-PDA) system and combines ultrasonic rapid extraction of saponins technology, which greatly saves detection time. The sample pretreatment time plus the detection time is a total of 20 minutes, which is much shorter than the 15 hours in the 2015 edition of the Pharmacopoeia. The overall operation is simple and suitable for popularization detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A chromatogram using a BEH HILIC 1.7uM 2.1X100mm column is shown;

[0018] Figure 2 Shows a chromatogram using an ACQUITY UPLCRHSS T3 1.8uM 2.1X100mm column;

[0019] Figure 3 A chromatogram using an ACQUITY UPLC@BEH-C18 column (1.7um×2.1×50mm) is shown;

[0020] Figure 4 A chromatogram using gradient elution 1) is shown;

[0021] Figure 5 A chromatogram using gradient elution 2) is shown;

[0022] Figure 6 A chromatogram using gradient elution 3) is shown;

[0023] Figure 7 A chromatogram using a detection wavelength of 193 nm is shown;

[0024] Figure 8 A chromatogram using a detection wavelength of 203 nm is shown;

[0025] Fig. 9 A chromatogram using a detection wavelength of 213 nm is shown;

[0026] Fig.10 A chromatogram using a detection wavelength of 223 nm is shown;

[0027] Fig.11 shows the chromatogram of 9 saponins determined by UPLC-PDA for the mixed standard solution 1;

[0028] Fig.12 shows the chromatogram of 9 saponins determined by UPLC-PDA for mixed standard solution 2;

[0029] Fig.13 The standard curve of notoginsenoside R1 is shown;

[0030] Fig.14 The standard curve of ginsenoside Rg1 is shown;

[0031] Fig.15 The standard curve of ginsenoside Re is shown;

[0032] Fig.16 The standard curve of ginsenoside Rb1 is shown;

[0033] Fig.17 The standard curve of ginsenoside Rc is shown;

[0034] Fig.18 The standard curve of ginsenoside Rg2 is shown;

[0035] Fig.19 The standard curve of ginsenoside Rb3 is shown;

[0036] Fig. 20 The standard curve of ginsenoside Rd is shown;

[0037] Fig.21 The standard curve of jujuboside B is shown;

[0038] Fig. 22 shows a chromatogram of a Panax notoginseng root sample; and

[0039] Fig.23 The chromatogram of the Panax notoginseng leaf sample is shown. DETAILED DESCRIPTION

[0040] The methods in the following embodiments are conventional methods unless otherwise specified.

[0041] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several changes and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0042] Establishment of a rapid quantitative detection method for saponins in Panax notoginseng

[0043] Standard curve preparation

[0044] 20 mg of saponin standards were accurately weighed (the saponin standards were: notoginsenoside R1, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, ginsenoside Rc, ginsenoside Rb3, ginsenoside Rd, ginsenoside Rg2, jujube saponin B), dissolved in 200 μL methanol, and prepared into a single standard stock solution, which was stored at -20°C. Jujube saponin B, which has similar properties to notoginsenosides, was used as an internal standard to ensure the accuracy of the established rapid quantitative detection method for saponins in notoginseng between batches.

[0045] Concentration calculation formula: c = m * B / V (c represents solution concentration; m represents standard mass; B represents standard purity; V represents solvent volume). Then the stock solution is diluted to a series of standard curve working solutions of 1000 μg / mL, 400 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, and 12.5 μg / mL in sequence. Jujube saponin B is used as the internal standard, and the preparation of the standard curve working solution is repeated in the above method.

[0046] Mixed standard solution

[0047] Notoginsenoside R1, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, ginsenoside Rc, ginsenoside Rb3, ginsenoside Rd, ginsenoside Rg2, and jujube saponin B were accurately weighed respectively, and all of them were dissolved in 200 μL of methanol to prepare a mixed standard solution 1 in which the concentration of each saponin standard was 400 μg / mL.

[0048] Accurately weigh notoginsenoside R1, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, ginsenoside Rc, ginsenoside Rb3, ginsenoside Rd, ginsenoside Rg2, and jujube saponin B respectively, and dissolve them all in 200 μL of methanol to prepare a mixed standard solution 2 in which the concentration of each saponin standard is 25 μg / mL.

[0049] Instrumental methods

[0050] The chromatographic column, elution conditions and detection conditions were optimized using 400 μg / mL mixed standard solution 1 as the sample.

[0051] The instrument used was Waters ACQUITY UPLC. The chromatographic columns used were: ACQUITY UPLC@BEH-C18 chromatographic column (1.7um×2.1×50mm), BEH HILIC 1.7uM2.1X100mm and ACQUITY UPLCRHSS T3 1.8uM2.1X100mm. Mobile phase A: acetonitrile, mobile phase B: water, flow rate 0.25mL / min. The sample volume was 2uL. The gradient elution was 1) 0-7min, 20%A; 7-13.5min, 20%A-60%A; 2) 0-6min, 20%A; 6-13min, 20%A-50%A; 3) 0-5min, 20%A; 5-10min, 20%A-45%A. The column temperature was 30°C, and the acquisition wavelengths of the photodiode array detector (PDA) were 193 nm, 203 nm, 213 nm, and 223 nm, respectively. The resolution was 1.2 nm, and the sampling rate was 10 pt / s.

[0052] Chromatographic columns

[0053] No signal was generated using the BEH HILIC 1.7uM 2.1X100mm column ( Figure 1 ). Using ACQUITY UPLCRHSS T3 1.8uM 2.1X100mm column, ginsenoside Rg1 and ginsenoside Re were not distinguished, and other peaks were tailing ( Figure 2 Using ACQUITY UPLC@BEH-C18 column (1.7um×2.1×50mm), ginsenoside Rg1 and ginsenoside Re were basically separated, while ginsenoside Rc and ginsenoside Rg2 were not well separated ( Figure 3 ), but in real samples, ginsenoside Rc is mainly present in the stems and leaves of Panax notoginseng, while ginsenoside Rg2 is mainly in the roots of Panax notoginseng, which does not affect the detection. Therefore, the BEH-C18 column was selected for the rapid quantitative detection method of saponins in Panax notoginseng.

[0054] Elution conditions

[0055] Using gradient elution condition 1 (0-7 min, 20% A; 7-13.5 min, 20% A-60% A), the following five saponins showed overlapping peaks ( Figure 4 ). Gradient elution condition 2 (0-6 min, 20% A; 6-13 min, 20% A-50% A) ( Figure 5 ); Ginsenoside Rb3 and ginsenoside Rd could not be distinguished. Gradient elution condition 3 (0-5min, 20% A; 5-10min, 20% A-45% A) was used ( Figure 6); 8 kinds of saponins were basically separated. Therefore, gradient elution condition 3 was selected for the rapid quantitative detection method of saponins in Panax notoginseng.

[0056] Detection wavelength

[0057] At four detection wavelengths 193nm ( Figure 7 )、203nm( Figure 8 )、213nm( Fig. 9 )、223nm( Fig.10 ), it was found that the waveform was stable and the peak height was suitable at 213nm wavelength. Therefore, the 213nm detection wavelength was selected for the rapid quantitative detection method of saponins in Panax notoginseng.

[0058] The instrumental method for rapid quantitative detection of saponins in Panax notoginseng was determined through optimization as follows:

[0059] The instrument used was Waters ACQUITY UPLC; the chromatographic column was ACQUITY UPLC@BEH-C18 column (1.7um×2.1×50mm); mobile phase A: acetonitrile, mobile phase B: water; flow rate 0.25mL / min; sample volume 2uL; gradient elution: 0-5min, 20%A; 5-10min, 20%A-45%A; column temperature 30°C; the collection wavelengths of the photodiode array detector (PDA) were 213nm; the separation was 1.2nm; and the sampling rate was 10pt / s.

[0060] result

[0061] The chromatogram of the rapid quantitative detection of the mixed standard solution 1 using the optimized conditions is as follows: Fig.11 Further, the chromatogram of the rapid quantitative detection of the mixed standard solution 2 as a sample using the optimized conditions is shown in Fig.12 The results showed that ginsenoside Rg1 and ginsenoside Re basically achieved peak baseline separation, and although ginsenosides Rc, Rg2 and Rb3 did not completely achieve baseline separation, Rc and Rb3 were mainly present in the aerial part of Panax notoginseng, and were basically not contained in the underground part, while Rg2 was mainly present in the underground part of Panax notoginseng, and was basically not contained in the aerial part, and the experimental verification basically met the requirements.

[0062] The standard curves of 9 saponins are shown in Figure 2. Figure 13-21 As shown in the figure, 8 of the 9 saponins detected were saponins contained in Panax notoginseng, and 1 exogenous saponin, jujuboside B, was used as an internal standard. The standard curve had good linearity, R 2All of them reached above 0.999. At the same time, the detection limit LOD and quantification limit LOQ values ​​were determined according to the blank background + 3*S / N and 10*S / N. The theoretical values ​​were verified (n=6), the unstable concentration was adjusted, and the actual detection limits and quantification limits of 9 standard saponins were determined.

[0063] The standard curves, linear ranges, detection limits, quantitation limits and sample detection repeatability of the nine saponins are shown in Figure 2.

[0064] As shown in Table 1.

[0065]

[0066]

[0067] Table 1

[0068] Note: DS is the above-ground part of Panax notoginseng, and DX is the underground part of Panax notoginseng.

[0069] Detection of above-ground and underground parts of Panax notoginseng

[0070] Panax notoginseng samples were collected from different Panax notoginseng planting areas in Yunnan Province using a three-point sampling method. A total of 20-30 plants were collected from each sampling point. The samples were cleaned, dried in an oven at 60°C to constant weight, and divided into the above-ground part (leaves) and underground part (roots) of Panax notoginseng, and tested using the rapid quantitative detection method for saponins in Panax notoginseng.

[0071] At the same time, jujuboside B was added to the samples of the above-ground and underground parts of Panax notoginseng for recovery experiments: 0.2 g of the above-ground and underground parts of Panax notoginseng were weighed respectively (accurate to mg), 5.0 mL of methanol was added, an appropriate amount of exogenous jujuboside B was added, the mixture was shaken and mixed, ultrasonically treated for 10 min, centrifuged at 8000 rpm / min for 5 min, 1 mL of the supernatant was filtered through a 0.22 uM filter membrane, and set aside.

[0072] The rapid quantitative detection method of saponins in Panax notoginseng was used to detect that Panax notoginseng root mainly contains six kinds of saponins: ginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rg2, ginsenoside Rb1 and ginsenoside Rd. Fig. 22 ); Panax notoginseng leaves mainly contain three saponins: ginsenoside Rb1, ginsenoside Rc and ginsenoside Rb3 ( Fig.23 ).

[0073] The stability of the established rapid quantitative detection method for saponins in Panax notoginseng was determined by conducting repeated experiments on samples of the above-ground and underground parts of Panax notoginseng (n=6). The analysis results showed that the RSD% was less than 10%.

[0074] The recoveries of the above-ground and underground parts were 93.19% and 99.37%, respectively, indicating that the established method for rapid quantitative detection of saponins in Panax notoginseng is accurate and stable, and can be used to detect the types and contents of 8 saponins in Panax notoginseng samples.

[0075] Example 1

[0076] Panax notoginseng sample testing

[0077] In July 2019, Panax notoginseng samples were collected from different Panax notoginseng planting areas in Yunnan Province using the three-point sampling method. A total of 20-30 plants were collected from each sampling point, cleaned, and dried in an oven at 60°C to constant weight. They were divided into the above-ground parts of Panax notoginseng (stems and leaves, referred to as DS) and the underground parts of Panax notoginseng (cut ends, taproots and fibrous roots, referred to as DX), and tested using the rapid quantitative detection method for saponins in Panax notoginseng.

[0078] The results are shown in Table 2, which show the differences in the types and contents of saponins in different parts of Panax notoginseng, which can well represent the quality characteristics of Panax notoginseng and are consistent with the quality requirements of Panax notoginseng in the 2020 edition of the Pharmacopoeia.

[0079] Table 2

[0080]

[0081]

[0082] Note: DS is the above-ground part of Panax notoginseng, and DX is the underground part of Panax notoginseng.

Claims

1. A rapid quantitative detection method for saponins in Panax notoginseng, comprising: Extract the Panax notoginseng sample with methanol to obtain a methanol extract of the Panax notoginseng sample; Ultrasonic treatment was performed when extracting Panax notoginseng samples with methanol; as well as The methanol extract of the Panax notoginseng sample is analyzed by using an ultra-high performance liquid chromatography-diode array detector system; The ultra-high performance liquid chromatography uses an ACQUITY UPLC@BEH-C18 1.7um×2.1×50mm chromatographic column; the ultra-high performance liquid chromatography conditions are as follows: mobile phase A: acetonitrile, mobile phase B: water; gradient elution: 0-5 min, 20% A; 5-10 min, 20%A-45%A; the acquisition wavelength of the diode array detector is 213 nm; the flow rate of the mobile phase is 0.25 mL / min; the column temperature of the chromatographic column is 30°C; The photoelectric separation of the diode array detector is 1.2 nm and the sampling rate is 10 pt / s; Jujuboside B is used as an internal standard in the ultra-high performance liquid chromatography; The saponins in the Panax notoginseng are notoginseng saponin R1, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, ginsenoside Rc, ginsenoside Rb3, ginsenoside Rd and ginsenoside Rg2.

2. The method according to claim 1, characterized in that The ultra-high performance liquid chromatography used a standard curve of notoginsenoside R1, a standard curve of ginsenoside Rg1, a standard curve of ginsenoside Rb1, a standard curve of ginsenoside Re, a standard curve of ginsenoside Rc, a standard curve of ginsenoside Rb3, a standard curve of ginsenoside Rd and a standard curve of ginsenoside Rg2.

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