A method for rapid determination of chlorogenic acid isomers in Polygonum villosa and its application

Abstract

The present invention belongs to the field of Chinese medicinal material analysis, and discloses a method for rapidly determining the content of chlorogenic acid isomers in Polygonum villosa, comprising the following steps: (1) preparation of a test solution; (2) preparation of a reference solution; (3) high performance liquid chromatography analysis and detection. The present invention also discloses the application of this method in identifying isomers in Polygonum villosa or determining the content of isomers in Polygonum villosa. The present invention clarifies for the first time that two isomers of chlorogenic acid exist simultaneously in Polygonum villosa: neochlorogenic acid and cryptochlorogenic acid, and can accurately determine their content in Polygonum villosa, providing an important reference basis for improving the quality control standards of Polygonum villosa. At the same time, the detection method of the present invention is simple to operate, rapid and efficient, highly sensitive, repeatable, stable and reliable, and can complete the detection of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid within 7 minutes, with good separation.

Description

Technical Field

[0001] The present invention belongs to the field of Chinese medicinal material analysis, and specifically, the present invention relates to a method for rapidly determining the content of chlorogenic acid isomers in Polygonum villosa by using high performance liquid chromatography, and application of the method in identifying isomers in Polygonum villosa or determining the content of isomers in Polygonum villosa. Background Art

[0002] Polygonum viviparum L, also known as: mountain sorghum, scorpion seven, monkey baby seven, scissors seven, dye cloth, is a perennial herbaceous plant of the Polygonaceae family. It belongs to the traditional Tibetan medicine. It is recorded as "Ranbu" in the "Jingzhu Materia Medica". Its rhizome is used as medicine. Polygonum viviparum grows in gullies or wet grasslands in high mountain areas, river beaches and forest edge shrubs. It is distributed in Tibet, Qinghai, Xinjiang, Inner Mongolia, Gansu, Sichuan and other places. It has the effects of antidiarrhea, antipyretic, blood stasis and hemostasis. Modern research shows that the main chemical components of Polygonum viviparum are tannins, volatile oils, polysaccharides, flavonoids, gallic acid, chlorogenic acid, etc., among which organic acid components are one of its main active substances.

[0003] The "Standard Tibetan Medicines of the Ministry of Health of the People's Republic of China (Volume 1)" only provides microscopic identification of Polygonum villosa medicinal materials, without any content determination items, and lacks a complete quality control standard. In the literature reports, gallic acid, β-sitosterol or flavonoids in Polygonum villosa are mostly detected. For example, the document "Research on the Quality Standard of Tibetan Medicinal Material Polygonum villosa" (Analytical Laboratory, 2008, 27 (S1): 100-103) used RP-HPLC to determine the content of quercetin in Polygonum villosa and thin layer chromatography to identify β-sitosterol; the document "Determination of Gallic Acid in Scorpion Seven by High Performance Liquid Chromatography" (China Pharmacy, 2005, 16 (10): 780-781) determined the content of gallic acid in Polygonum villosa; the document "Research on Quality Control Method of Scorpion Seven" (Journal of Pharmaceutical Analysis, 2005, 25 (11): 1343-1345) used thin layer chromatography to identify gallic acid in Scorpion Seven and used high performance liquid chromatography to quantify it. The literature "Determination of chlorogenic acid content in Panax notoginseng and its application of methodology" (Ningxia Medical Journal, 2011, 33(1): 6-7), "HPLC method for simultaneous determination of gallic acid, chlorogenic acid and quercetin content in Polygonum villosa" (Journal of Ningxia Normal University (Natural Science), 2014, 35(3): 74-77) and application number CN201610842261.7, invention name "A method for determining organic acids and flavonoids in Polygonum villosa" all determined the content of chlorogenic acid in Polygonum villosa, but did not mention chlorogenic acid isomers. The literature "HPLC-MS analysis of phenolic components in Polygonum villosa" (Today's Pharmacy, 2018(01): 31-34) detected the simultaneous presence of chlorogenic acid isomers in Polygonum villosa, but failed to identify what substance it was and did not conduct quantitative analysis. Chlorogenic acid is one of the main active ingredients in Polygonum villosa. There are few methods for determining the chlorogenic acid content in Polygonum villosa in current research reports, and the preparation and analysis time of the test samples are relatively long. No reports on the determination of the chlorogenic acid isomers in Polygonum villosa have been found, and the quality control of this medicinal material has not been effectively carried out. Summary of the invention

[0004] The invention provides a method for rapidly determining chlorogenic acid isomers in Polygonum villosa, and also discloses the application of the method in identifying isomers in Polygonum villosa or determining the content of isomers in Polygonum villosa.

[0005] The present invention is achieved through the following technical solutions:

[0006] In one aspect, the present invention provides a method for rapidly determining chlorogenic acid isomers in Polygonum villosa, characterized in that the method comprises the following steps:

[0007] (1) Preparation of test solution: Extract Polygonum villosa with 50% methanol to prepare the test solution;

[0008] (2) Preparation of reference solution:

[0009] Chlorogenic acid reference solution: Add methanol to chlorogenic acid to prepare chlorogenic acid reference solution;

[0010] Neochlorogenic acid reference solution: Take neochlorogenic acid and add methanol to prepare neochlorogenic acid reference solution;

[0011] Chlorogenic acid reference solution: Add methanol to chlorogenic acid to prepare chlorogenic acid reference solution;

[0012] Mixed reference solution: Take the reference substances of chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid and add methanol to prepare a mixed reference solution of chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid;

[0013] (3) The test solution in step (1) and the reference solution in step (2) were tested and analyzed according to the following high performance liquid chromatography detection conditions: C18 chromatographic column, mobile phase 0.1% acetic acid (A)-methanol (B), gradient elution, flow rate 0.8-1.5 mL / min; column temperature 25-35° C.; detection wavelength 330 nm.

[0014] In some embodiments, the preparation step of the test solution includes: using 50% methanol to ultrasonically or vortex extract the Polygonum villosa, centrifuging, and filtering the supernatant to obtain the test solution.

[0015] In some embodiments, the solid-liquid ratio of the Polygonum villosa to the 50% methanol is 1:10 to 1:50 g / mL, such as 1:10, 1:30, 1:50 g / mL, preferably 1:30 g / mL.

[0016] In some embodiments, the ultrasound time is 1 to 45 min, such as 1, 3, 5, 25, or 45 min, preferably 1 to 5 min, and more preferably 5 min.

[0017] In some embodiments, the vortex time is 1 to 5 min, such as 1, 3, or 5 min, preferably 3 to 5 min, and more preferably 5 min.

[0018] In some embodiments, the centrifugation time is 3-10 min, preferably 5 min.

[0019] In some embodiments, the centrifugal speed is 3000-8000 rpm / min, preferably 5000 rpm / min.

[0020] In some embodiments, the preparation steps of the test solution include: the Polygonum villosa is the rhizome of Polygonum villosa, accurately weighing 0.25 g of the Polygonum villosa rhizome powder that has passed through a No. 3 sieve, ultrasonically or vortex-extracting it in 7.5 mL of 50% methanol for 5 minutes, taking it out and cooling it, weighing it, making up the lost weight with 50% methanol, shaking it well, centrifuging it at 5000 rpm / min for 5 minutes, taking the supernatant and passing it through a 0.22 μm organic filter membrane, and taking the filtrate to obtain the test solution.

[0021] In some embodiments, the C18 chromatographic column is Agilent PoroShell 120EC-C18 (50 mm×4.6 mm, 2.7 μm) or Agilent ZORBAX SB-C18 (150 mm×4.6 mm, 5 μm), preferably Agilent PoroShell 120EC-C18 (50 mm×4.6 mm, 2.7 μm).

[0022] In some embodiments, the C18 chromatographic column is Agilent PoroShell 120EC-C18 (50 mm × 4.6 mm, 2.7 μm), with a flow rate of 1 mL·min -1 , column temperature 35°C, gradient elution conditions are as follows:

[0023] 0-12min, B increases from 10% to 11%-15%; such as 0-12min, B increases from 10% to 11%; such as 0-12min, B increases from 10% to 12%; such as 0-12min, B increases from 10% to 15%; or

[0024] 0-7min, B increased from 10% to 11%~15%, 7-12min, B increased from 11%~15% to 100%; such as 0-7min, B increased from 10% to 11%, 7-12min, B increased from 11% to 100%; such as 0-7min, B increased from 10% to 13%, 7-12min, B increased from 13% to 100%; such as 0-7min, B increased from 10% to 15%, 7-12min, B increased from 15% to 100%;

[0025] Preferably, B increases from 10% to 13% in 0-7 min, and from 13% to 100% in 7-12 min.

[0026] In some embodiments, the C18 chromatographic column is Agilent PoroShell 120EC-C18 (50 mm×4.6 mm, 2.7 μm); the gradient elution conditions are: 0-7 min, B increases from 10% to 13%, 7-12 min, B increases from 13% to 100%; flow rate 0.8-1.5 mL·min -1; Column temperature 35°C; Detection wavelength 330nm; Injection volume 1-2.5μL, preferably, the injection volume is 2μL.

[0027] On the other hand, the present invention also discloses the application of the above method in identifying isomers in Polygonum villosa or determining the content of isomers in Polygonum villosa.

[0028] The method for rapidly determining the content of chlorogenic acid isomers in Polygonum villosa described in the present invention clarifies for the first time that two isomers of chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid, exist simultaneously in Polygonum villosa, and can accurately determine the content of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid in Polygonum villosa, providing an important reference basis for improving the quality control standards of Polygonum villosa.

[0029] The present invention creatively discovered in the process of separating the isomers of chlorogenic acid that although the polarities of the three organic acids are not much different, the separation degree between chlorogenic acid and the subsequent impurity peak does not meet the requirements when conventional chromatographic elution conditions such as isocratic 15% methanol, 13% methanol, etc. are used for elution. When the mobile phase is changed to acetonitrile with similar polarity for elution, the three chromatographic peaks cannot achieve baseline separation. However, when the chromatographic conditions of the present invention are used for analysis, it can be ensured that the three organic acid components achieve baseline separation, and the chromatographic peaks are correspondingly higher.

[0030] At the same time, the method for preparing chlorogenic acid components provided by the present invention is simple and fast, and the extraction can be completed in 5 minutes, which greatly saves time. In addition, the method for high performance liquid chromatography analysis and detection of the present invention is simple to operate, fast and efficient, highly sensitive, reproducible, stable and reliable, and can complete the detection of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid within 7 minutes, and the separation is good.

[0031] Definition of terms

[0032] In the description of this specification, regardless of whether the words "about" or "approximately" are used, all the numbers disclosed herein are approximate values. The value of each number may differ by 1%, 2%, or 5%.

[0033] In the description of this specification, the percentages expressing the contents of the solvents used are all volume percentages. For example, "50% methanol" means that the volume fraction of methanol in the methanol aqueous solution is 50%; for example, "0.1% acetic acid (A)-methanol (B)" means that the volume fraction of acetic acid in the 0.1% acetic acid aqueous solution is 0.1%.

[0034] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a chromatogram of the test solution of the Polygonum villosa rhizome powder S1 in Example 1 of the present invention in the gradient elution of Agilent ZORBAX SB-C18, Figure 1 A is the chromatogram of gradient elution 1-1, Figure 1 B is the chromatogram of gradient elution 1-2, Figure 1 C is the chromatogram of gradient elution 1-3, Figure 1 D is a chromatogram of gradient elution 1-4; wherein peak 1, peak 2, and peak 3 in the chromatogram represent neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid, respectively, the same below; min represents minutes, the same below;

[0036] Figure 2 is a chromatogram of the test solution of the Polygonum villosa rhizome powder S1 in Example 1 of the present invention in the gradient elution of Agilent PoroShell120EC-C18, Figure 2 A is the chromatogram of gradient elution 2-1, Figure 2 B is the chromatogram of gradient elution 2-2, Figure 2 C is the chromatogram of gradient elution 2-3, Figure 2 D is the chromatogram of gradient elution 2-4, Figure 2 E is the chromatogram of gradient elution 2-5, Figure 2 F is the chromatogram of gradient elution 2-6;

[0037] Figure 3 It is a chromatogram of the mixed reference solution of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid in Example 1 of the present invention at gradient elution 2-5;

[0038] Figure 4 is a chromatogram of the test solution of the Polygonum villosa rhizome powder S1 in Example 2 of the present invention at flow rates of 0.8 mL / min, 1.0 mL / min, 1.2 mL / min, and 1.5 mL / min, respectively;

[0039] Figure 5 is a chromatogram of the test solution of the Polygonum villosa rhizome powder S1 in Example 3 of the present invention when the column temperatures are set to 25° C., 30° C., 35° C., 40° C., and 45° C.;

[0040] Figure 6 is a chromatogram of a test solution of the Polygonum villosa rhizome powder S1 in Example 4 of the present invention in a mobile phase of 0.1% acetic acid (A)-acetonitrile (B);

[0041] Figure 7 is a chromatogram of the test solution of the Polygonum villosa rhizome powder S1 in Example 4 of the present invention in a mobile phase of ultrapure water (A)-methanol (B);

[0042] Figure 8 is a chromatogram of a test solution of the Polygonum villosa rhizome powder S1 in Example 4 of the present invention in a mobile phase of 0.2% acetic acid (A)-methanol (B);

[0043] Fig. 9 It is a chromatogram of the test solution of the Polygonum villosa rhizome powder S1 in Example 4 of the present invention in a mobile phase of 0.1% formic acid (A)-methanol (B). DETAILED DESCRIPTION

[0044] Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0045] The present invention is further explained below with reference to specific embodiments.

[0046] Example

[0047] 1. Instrument

[0048] Agilent 1260 high performance liquid chromatograph, UV-visible spectrophotometer (Agilent Technologies, Inc., USA); chromatographic columns (Agilent PoroShell 120EC-C18 (50 mm × 4.6 mm, 2.7 μm), Agilent ZORBAX SB-C18 (150 mm × 4.6 mm, 5 μm)); XS 205 1 / 100000 electronic analytical balance (Mettler-Toledo Instrument Co., Ltd., USA); Milli-Q ultrapure water system (Merck Millipore, Germany); SBL-22DT ultrasonic instrument (Ningbo Xinzhi Biotechnology Co., Ltd.); Sorvall ST40R desktop centrifuge (Thermo Fisher Scientific); Tube-Mill 100 grinder (IKA, Germany); vortex mixer Vortex Geniius 3 (IKA, Germany).

[0049] 2. Drug testing

[0050] Methanol (analytical grade, Chengdu Kelong Reagent Co., Ltd., batch number: 190917 1), glacial acetic acid (chromatographic grade, Aladdin Shanghai Co., Ltd., batch number: 180108 1), formic acid (chromatographic grade, Aladdin Shanghai Co., Ltd., batch number: D1919032), acetonitrile (chromatographic grade, Spectrum, batch number: 3IL0008), methanol (chromatographic grade, Spectrum, batch number: 3IL0001), neochlorogenic acid (purity 99.9%, Shanghai Yuanye Biotechnology Co., Ltd., batch number: P27A10L87091), chlorogenic acid (purity 98.9%, Shanghai Yuanye Biotechnology Co., Ltd., batch number: Y22M8K36544), cryptochlorogenic acid (purity 98.6%, Shanghai Yuanye Biotechnology Co., Ltd., batch number: P11M10L88123), water (ultrapure water).

[0051] Test samples: 12 batches of Polygonum villosa rhizome powder: S1-S3 (produced in Yunnan); S4-S7 (produced in Sichuan); S8-S10 (produced in Guizhou); S11-S12 (produced in Tibet).

[0052] Example 1 Chromatographic Conditions Elution Gradient Investigation

[0053] 1.1 Preparation of test solution

[0054] Accurately weigh about 0.25 g of Polygonum villosa rhizome powder S1 (passed through No. 3 sieve) and place it in a stoppered conical flask, accurately measure 7.5 mL of 50% methanol, weigh the weight, sonicate in an ultrasonic instrument for 5 minutes, take out and cool, weigh the weight, make up the lost weight with 50% methanol, shake well, centrifuge at 5000 rpm / min for 5 minutes, take the supernatant and pass it through a 0.22 μm organic filter membrane, take the filtrate to obtain the test solution.

[0055] 1.2 Preparation of reference solution

[0056] Accurately weigh appropriate amounts of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid into volumetric flasks, add methanol solution, and prepare concentrations of 0.6194 mg / mL neochlorogenic acid, 0.4802 mg / mL chlorogenic acid, and 1.2093 mg / mL cryptochlorogenic acid, respectively.

[0057] Accurately measure appropriate amounts of the above-prepared neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid reference solutions into volumetric flasks, and add methanol solution to prepare mixed reference solutions of appropriate concentrations.

[0058] 1.3 Chromatographic conditions

[0059] (1) The chromatographic column was Agilent ZORBAX SB-C18 (150 mm × 4.6 mm, 5 μm), the mobile phase was 0.1% acetic acid (A)-methanol (B), and the flow rate was 1 mL min -1 , detection wavelength: 330nm, column temperature: 35℃; injection volume: 2μL, gradient elution conditions:

[0060] ① Gradient elution 1-1: 0-30min, B increased from 10% to 13%, 30-35min, B increased from 13% to 100%, 35-37min, 100% B, 37-40min, B decreased from 100% to 10%. The chromatographic results are as follows Figure 1 -A;

[0061] ② Gradient elution 1-2: 0-30min, B increased from 10% to 15%, 30-35min, B increased from 15% to 100%, 35-37min, 100% B, 37-40min, B decreased from 100% to 10%. The chromatographic results are as follows Figure 1 -B;

[0062] ③ Gradient elution 1-3: 0-20min, B increased from 10% to 15%, 20-25min, B increased from 15% to 100%, 25-27min, 100% B, 27-30min, B decreased from 100% to 10%. The chromatographic results are as follows Figure 1 -C;

[0063] ④ Gradient elution 1-4: 0-15min, 15% B, 15-20min, B increased from 15% to 100%, 20-22min, 100% B; 22-25min, B decreased from 100% to 15%. The chromatographic results are as follows Figure 1 -D.

[0064] (2) The chromatographic column was Agilent PoroShell 120EC-C18 (50 mm × 4.6 mm, 2.7 μm), the mobile phase was 0.1% acetic acid (A)-methanol (B), and the flow rate was 1 mL min -1 , detection wavelength: 330nm, column temperature: 35℃; injection volume: 2μL, gradient elution conditions:

[0065] ① Gradient elution 2-1: 0-12min, B increased from 10% to 15%, 12-15min, B increased from 15% to 100%, 15-18min, 100% B, 18-20min, B decreased from 100% to 10%. The chromatographic results of the test sample are as follows Figure 2 -A;

[0066] ② Gradient elution 2-2: 0-12min, B increased from 10% to 12%, 12-15min, B increased from 12% to 100%, 15-18min, 100% B, 18-20min, B decreased from 100% to 10%. The chromatographic results of the test sample are as follows Figure 2 -B;

[0067] ③ Gradient elution 2-3: 0-12min, B increased from 10% to 11%, 12-15min, B increased from 11% to 100%, 15-18min, 100% B, 18-20min, B decreased from 100% to 10%. The chromatographic results of the test sample are as follows Figure 2 -C;

[0068] ④ Gradient elution 2-4: 0-7min, B increased from 10% to 15%, 7-12min, B increased from 15% to 100%, 12-15min, B decreased from 100% to 10%. The chromatographic results of the test sample are as follows Figure 2 -D;

[0069] ⑤ Gradient elution 2-5: 0-7min, B increased from 10% to 13%, 7-12min, B increased from 13% to 100%, 12-15min, B decreased from 100% to 10%. The chromatographic results of the test sample are as follows Figure 2 -E, the chromatographic results of the mixed reference substance are as follows Figure 3 ;

[0070] ⑥ Gradient elution 2-6: 0-7min, B increased from 10% to 11%, 7-12min, B increased from 11% to 100%, 12-15min, B decreased from 100% to 10%. The chromatographic results of the test sample are as follows Figure 2 -F.

[0071] 1.4 Experimental Results

[0072] Depend on Figure 1 It can be seen that when the chromatographic column Agilent ZORBAX SB-C18 (150mm×4.6mm, 5μm) was used and the elution conditions were 1-1 to 1-3, the separation of each peak met the requirements, but the elution time was long. Increasing the proportion of the organic phase can shorten the elution time. Therefore, when the elution condition 1-4 was directly changed to 15% methanol isocratic elution, although the elution time of the target peak of chlorogenic acid was shortened by 6min compared with the elution condition 1-3, the separation of chlorogenic acid and the subsequent peak was poor ( Figure 1 The resolutions of chlorogenic acid and the latter peak in AD were 2.40, 2.39, 2.19, and 1.56, respectively).

[0073] Depend on Figure 2Results A to C show that when the chromatographic column Agilent PoroShell 120EC-C18 (50 mm × 4.6 mm, 2.7 μm) was used and the elution condition was 0 to 12 min, increasing the proportion of methanol in the eluent had little effect on the elution time and peak shape of each peak ( Figure 2 The resolutions of chlorogenic acid and the latter peak in A to C were 1.94, 2.03, and 2.01, respectively); Figure 2 Results from D to F show that as the proportion of methanol increases from 0 to 7 min, the peak time of cryptochlorogenic acid gradually shortens, while it has no significant effect on the peak time of neochlorogenic acid and chlorogenic acid ( Figure 2 The separation degrees of chlorogenic acid and the latter peak in D~F are 1.95, 1.98 and 1.97 respectively); the peak shape of chlorogenic acid under elution condition 2-4 is slightly tailing, which is worse than that under elution conditions 2-5 and 2-6; the peaks under elution conditions 2-1~2-6 are well separated. Since the analysis time of elution 2-5 is shorter and the peak shape is more symmetrical, elution condition 2-5 is preferred after comprehensive consideration.

[0074] Example 2 Investigation of Chromatographic Conditions and Flow Rates

[0075] According to the method under 1.1 of Example 1, the rhizome powder of Polygonum villosa (S1) was accurately weighed to prepare the test solution, and the sample was injected according to the chromatographic conditions 2-5 under 1.3 of Example 1, wherein the flow rates in the chromatographic conditions were set to 0.8 mL / min, 1.0 mL / min, 1.2 mL / min, and 1.5 mL / min, respectively. The results are as follows: Figure 4 shown.

[0076] Depend on Figure 4 It can be seen that with the increase of flow rate, the elution time of each peak is advanced; the flow rate is 0.8mL / min (the separation degree between the latter peak and the chlorogenic acid peak is 1.98), 1.0mL / min (the separation degree between the latter peak and the chlorogenic acid peak is 1.96), 1.2mL / min (the separation degree between the latter peak and the chlorogenic acid peak is 1.89), 1.5mL / min (the separation degree between the latter peak and the chlorogenic acid peak is 1.81), the separation degree of each peak can meet the requirements (R>1.5), based on the peak shape and elution time, the preferred flow rate is 1.0mL / min.

[0077] Example 3 Investigation of Chromatographic Conditions and Column Temperature

[0078] According to the method under 1.1 of Example 1, the rhizome powder of Polygonum villosa (S1) was accurately weighed to prepare a test solution, and the sample was injected according to the chromatographic conditions 2-5 under 1.3 of Example 1, wherein the column temperatures in the chromatographic conditions were set to 25°C, 30°C, 35°C, 40°C, and 45°C, respectively. The results are as follows: Figure 5 What I see.

[0079] Depend on Figure 5It can be seen that as the column temperature increases, the elution time of each peak is advanced, chlorogenic acid and the subsequent peak are separated as the temperature increases, while cryptochlorogenic acid gradually approaches the previous peak. When the column temperature is 40°C and 45°C, the separation effect is not good. In summary, in order to ensure the separation effect of chlorogenic acid and the subsequent peak, cryptochlorogenic acid and the previous peak, the column temperature is selected to be 35°C.

[0080] Example 4 Investigation of Chromatographic Conditions Mobile Phase

[0081] According to the method under 1.1 of Example 1, the rhizome powder of Polygonum villosa (S1) was accurately weighed to prepare a test solution, and the sample was injected according to the chromatographic conditions 2-5 under 1.3 of Example 1, wherein the mobile phases in the chromatographic conditions were respectively set to: 0.1% acetic acid (A)-acetonitrile (B), ultrapure water (A)-methanol (B), 0.2% acetic acid (A)-methanol (B), 0.1% formic acid (A)-methanol (B), and the results were as follows: Figure 6 , Figure 7 , Figure 8 , Fig. 9 shown.

[0082] Depend on Figure 6 It can be seen that after the mobile phase B changes from methanol to acetonitrile, the elution capacity of the mobile phase increases and the separation of the peaks is poor. Figure 7 It can be seen that after the mobile phase A replaced 0.1% acetic acid with ultrapure water, the peak responses were lower. Figure 8 and Fig. 9 It can be seen that when the acidity of mobile phase A is increased and 0.1% acetic acid is replaced with 0.2% acetic acid or 0.1% formic acid, the separation of chlorogenic acid and the latter peak is poor, so 0.1% acetic acid is more suitable for mobile phase A.

[0083] Example 5 Investigation of extraction solvent

[0084] According to the method under 1.1 in Example 1, accurately weigh the rhizome powder of Polygonum villosa (S1) to prepare the test solution. According to the method under 1.2 in Example 1, prepare the reference solutions of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid, respectively, and dilute them to 0.06194 mg / ml, 0.09604 mg / ml, and 0.06047 mg / ml, respectively. The extraction solvents are set to ultrapure water, 50% methanol, and anhydrous methanol, respectively. The obtained test solution and reference substance dilution are injected according to the chromatographic conditions 2-5 under 1.3 in Example 1. Parallel operations are performed to examine the effect of the extraction solvent on the extraction results.

[0085] The results showed that when the extraction solvent was 50% methanol, the contents of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid were the highest, indicating that when the extraction solvent was 50% methanol, the sample extraction efficiency was the highest, the operation was parallel, and the results were reliable.

[0086] Example 6 Investigation of different ultrasound times

[0087] According to the method under 1.1 in Example 1, the rhizome powder of Polygonum villosa (S1) was accurately weighed to prepare the test solution in duplicate, wherein the ultrasonic time was set to 1min, 3min, 5min, 25min, and 45min, respectively. According to the method under 1.2 in Example 1, the reference solutions of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid were prepared respectively, and diluted to 0.06194mg / ml, 0.09604mg / ml, and 0.06047mg / ml, respectively. The obtained test solution and reference dilution were sampled and analyzed according to the chromatographic conditions 2-5 under 1.3 in Example 1. The effect of different ultrasonic times on the extraction results was investigated, and the results are shown in Table 1.

[0088] Table 1 Effect of different ultrasonic extraction times on the extraction efficiency of three organic acid components in Polygonum villosa (n=2, )

[0089]

[0090] As can be seen from Table 1, the contents of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid were slightly different at different ultrasonic times, indicating that the neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid in the rhizomes of Polygonum villosa can be completely extracted by ultrasonication for 1 min. Because the ultrasonication time of 1 min or 3 min is too short, a large amount of sample may lead to a large experimental error. The optimal ultrasonication time is 5 min.

[0091] Example 7 Investigation of different vortex times

[0092] Weigh about 0.25g of Polygonum villosa rhizome powder S1 (passed through No. 3 sieve) accurately and place it in a stoppered conical bottle in duplicate. Accurately measure 7.5mL of 50% methanol, weigh it, vortex it, take it out and let it cool, weigh it, make up the lost weight with 50% methanol, shake it well, centrifuge it at 5000rpm / min for 5min, take the supernatant and filter it through a 0.22μm organic filter membrane, take the filtrate, and get the test solution. The vortex time is set to 1min, 3min, and 5min respectively.

[0093] According to the method under 1.2 of Example 1, the reference solutions of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid were prepared and diluted to 0.06194 mg / ml, 0.09604 mg / ml, and 0.06047 mg / ml, respectively. The obtained test solution and reference dilution were injected and analyzed according to the chromatographic conditions 2-5 under 1.3 of Example 1. The effect of different vortex times on the extraction results was investigated, and the results are shown in Table 2.

[0094] Table 2 Effect of different vortexing times on the extraction efficiency of three organic acid components from Polygonum villosa (n=2, )

[0095]

[0096] It can be seen from Table 2 that the contents of the three components extracted by vortexing for 1 minute are lower than those by vortexing for 3 minutes or 5 minutes; after vortexing for 3 minutes or 5 minutes, the contents of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid are all slightly different, and compared with ultrasound for 5 minutes, the contents of the three components are all slightly different, indicating that the neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid in the rhizomes of Polygonum villosa can be completely extracted by vortexing for 3 minutes. The time of 3 minutes is too short, and a large amount of sample may lead to large experimental errors. The preferred vortexing time is 5 minutes.

[0097] Example 8 Investigation of different material-liquid ratios

[0098] According to the method under 1.1 in Example 1, the Polygonum villosa rhizome powder (S1) was accurately weighed to prepare the test solution in duplicate, wherein the solid-liquid ratio of the Polygonum villosa rhizome powder to the extraction solvent 50% methanol was set to 1:10, 1:30, and 1:50, respectively.

[0099] According to the method under 1.2 of Example 1, the reference solutions of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid were prepared and diluted to 0.06194 mg / ml, 0.09604 mg / ml and 0.06047 mg / ml, respectively. The obtained test solution and reference dilution were injected and analyzed according to the chromatographic conditions 2-5 under 1.3 of Example 1. The effect of the material-liquid ratio on the extraction efficiency was investigated.

[0100] The results showed that the contents of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid in the rhizomes of Polygonum villosa were similar at different solid-liquid ratios, indicating that different solid-liquid ratios had little effect on the extraction efficiency, the operation was parallel and the results were reliable. In order to obtain more test solution and save extraction solvent, the preferred solid-liquid ratio was 1:30, that is, about 0.25 g of Polygonum villosa rhizome powder S1 (passed through No. 3 sieve) was accurately weighed and 7.5 mL of 50% methanol was added.

[0101] Example 9 Methodological Investigation

[0102] The content determination method of the present invention is subjected to methodological validation, including precision test, repeatability test, stability test, sample recovery test, linear relationship and range.

[0103] 9.1 Precision test

[0104] According to the method under 1.1 in Example 1, the rhizome powder of Polygonum villosa (S1) was accurately weighed to prepare the test solution, and the sample was injected 6 times according to the chromatographic conditions 2-5 under 1.3 in Example 1, and the peak areas were recorded, and the RSD of the peak areas of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid was calculated. The results showed that the RSD of the peak areas of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid were 0.20%, 0.19%, and 0.17%, respectively, all less than 1%, indicating that the instrument had good precision.

[0105] 9.2 Repeatability test

[0106] ① Repeatability test of ultrasonic extraction

[0107] According to the method under 1.2 in Example 1, the reference solutions of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid were prepared respectively, and diluted to 0.06194 mg / ml, 0.09604 mg / ml, and 0.06047 mg / ml, respectively. According to the method under 1.1 in Example 1, 6 portions of the rhizome powder of Polygonum villosa (S1) were accurately weighed to prepare the test solution, and the sample was analyzed according to the chromatographic conditions of 2-5 under 1.3 in Example 1, the peak area was determined, and the contents of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid were calculated respectively. The results showed that the RSDs of the contents of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid were 0.79%, 0.86%, and 0.77%, respectively, all less than 1%, indicating that the method has good repeatability.

[0108] ② Vortex extraction repeatability test

[0109] According to the method under 1.2 in Example 1, the reference solutions of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid were prepared respectively, and diluted to 0.06194 mg / ml, 0.09604 mg / ml, and 0.06047 mg / ml, respectively. According to the method in Example 7, 6 portions of the rhizome powder of Polygonum villosa (S1) were accurately weighed to prepare the test solution, and the sample was analyzed according to the chromatographic conditions 2-5 under 1.3 in Example 1, and the peak area was determined, and the contents of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid were calculated respectively. The results showed that the RSDs of the contents of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid were 0.65%, 0.56%, and 0.67%, respectively, all less than 1%, indicating that the method had good repeatability.

[0110] 9.3 Stability test

[0111] According to the method under 1.1 in Example 1, the rhizome powder of Polygonum villosa (S1) was accurately weighed to prepare the test solution. According to the chromatographic conditions of 2-5 under 1.3 in Example 1, the sample was injected once every 2 hours within 0 to 12 hours, the peak area was determined, and the RSD of the peak areas of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid was calculated. The results showed that the RSD of the peak areas of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid were 0.22%, 0.25%, and 0.26%, respectively, which were all less than 1%, indicating that the three components of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid in Polygonum villosa were stable within 12 hours.

[0112] 9.4 Sample recovery test

[0113] Take 6 portions of Polygonum villosa rhizome powder (S1) with known contents of three components: neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid, 0.25 g per portion, accurately weigh and add a certain amount of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid reference substances, respectively, and prepare the test solution according to the method under 1.1 in Example 1. The sample is injected according to the chromatographic conditions 2-5 under 1.3 in Example 1, the peak area is determined, and the content and recovery rate of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid in the sample are calculated. The results showed that the recoveries of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid were all between 90% and 106% (neochlorogenic acid was 105.33%, chlorogenic acid was 100.15% and cryptochlorogenic acid was 105.60%), and the RSDs were 0.31%, 0.97% and 0.44%, respectively, all less than 1%, indicating that this method can accurately determine the contents of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid in Polygonum villosa.

[0114] 9.5 Linear Relationship and Range

[0115] According to the method under 1.2 of Example 1, the reference solutions of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid were prepared respectively, and diluted to 0.01239 mg / mL, 0.04802 mg / mL and 0.00967 mg / mL respectively. According to the chromatographic conditions of 2-5 under 1.3 of Example 1, 1 μL, 2 μL, 4 μL, 8 μL, 12 μL and 18 μL were injected respectively to determine the peak area. A linear review was performed with the peak area as the ordinate (Y) and the sample amount (unit: μg) as the abscissa (X), and the regression equation, correlation coefficient and linear range of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid were obtained. The results are shown in Table 3. As shown in Table 3, neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid are in the range of 0.0124-0.2230 μg, 0.0480-0.8644 μg, and 0.0097-0.1741 μg, respectively, and the linear relationship is good. The reference solution was diluted step by step, and then entered into the high performance liquid chromatograph for analysis according to the chromatographic conditions 2-5 under 1.3 of Example 1, and the concentration when the signal-to-noise ratio S / N≈10 was used as the quantitative limit of the component. The three components and quantitative limits were obtained, respectively, and the results are shown in Table 3.

[0116] Table 3 Linear relationship and range results of three organic acid components in Polygonum villosa

[0117]

[0118] Example 10 Content Determination

[0119] According to the method under 1.2 of Example 1, the reference solutions of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid were prepared respectively, and diluted to 0.06194 mg / ml, 0.09604 mg / ml and 0.06047 mg / ml respectively; the samples of Polygonum villosa (S1-S15) were taken, and the test solution was prepared according to the method under 1.1 of Example 1, and the analysis and detection were carried out according to the chromatographic conditions 2-5 under 1.3 of Example 1. The contents of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid in the Polygonum villosa samples were calculated by the external standard method, and the results are shown in Table 4.

[0120] Table 4 Results of content determination of Polygonum villosa samples (n=2, )

[0121]

[0122] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for rapidly determining the content of chlorogenic acid isomers in Polygonum villosa, characterized in that: The chlorogenic acid isomers include neochlorogenic acid and cryptochlorogenic acid; the method comprises the following steps: (1) Preparation of test solution: Extract Polygonum villosa with 50% methanol to prepare the test solution; (2) Preparation of reference solution: Chlorogenic acid reference solution: Add methanol to chlorogenic acid to prepare chlorogenic acid reference solution; Neochlorogenic acid reference solution: Take neochlorogenic acid and add methanol to prepare neochlorogenic acid reference solution; Chlorogenic acid reference solution: Chlorogenic acid is added with methanol to prepare chlorogenic acid reference solution; Mixed reference solution: Take the reference substances of chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid and add methanol to prepare a mixed reference solution of chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid; (3) The test solution in step (1) and the reference solution in step (2) were tested and analyzed according to the following high performance liquid chromatography detection conditions: C18 chromatographic column, mobile phase: mobile phase A is 0.1% acetic acid; mobile phase B is methanol, gradient elution; flow rate is 0.8-1.5 mL / min; column temperature is 25-35° C.; detection wavelength is 330 nm; The C18 chromatographic column is Agilent ZORBAX SB-C18, 150 mm × 4.6 mm, 5 μm, and the gradient elution conditions are as follows: ① Gradient elution 1-1: 0-30min, mobile phase B increased from 10% to 13%; 30-35min, mobile phase B increased from 13% to 100%; 35-37min, 100% mobile phase B; 37-40min, mobile phase B decreased from 100% to 10%; or ② Gradient elution 1-2: 0-30min, mobile phase B increased from 10% to 15%; 30-35min, mobile phase B increased from 15% to 100%; 35-37min, 100% mobile phase B; 37-40min, mobile phase B decreased from 100% to 10%; or ③ Gradient elution 1-3: 0-20min, mobile phase B increased from 10% to 15%; 20-25min, mobile phase B increased from 15% to 100%; 25-27min, 100% mobile phase B; 27-30min, mobile phase B decreased from 100% to 10%; or ④ Gradient elution 1-4: 0-15 min, 15% mobile phase B; 15-20 min, mobile phase B increased from 15% to 100%; 20-22 min, 100% mobile phase B; 22-25 min, mobile phase B decreased from 100% to 15%.

2. The method according to claim 1, characterized in that The preparation steps of the test solution include: extracting Polygonum villosa by ultrasonic or vortexing with 50% methanol, centrifuging, and filtering the supernatant to obtain the test solution.

3. The method according to any one of claims 1 to 2, characterized in that: The solid-liquid ratio of the Polygonum villosa to the 50% methanol is 1:10 to 1:50 g / mL.

4. The method according to any one of claims 1 to 2, characterized in that: The solid-liquid ratio of the Polygonum villosa to the 50% methanol is 1:30 g / mL.

5. The method according to claim 2, characterized in that: The ultrasound time is 1 to 45 minutes; The vortexing time is 1 to 5 minutes.

6. The method according to claim 2, characterized in that The ultrasonic time is 1 to 5 minutes.

7. The method according to claim 2, characterized in that: The ultrasonic time is 5 minutes.

8. The method according to claim 2, characterized in that: The vortexing time is 3 to 5 minutes.

9. The method according to claim 2, characterized in that: The vortex time was 5 min.

10. The method according to claim 2, characterized in that The centrifugation time is 3-10 min; the centrifugation speed is 3000-8000 rpm / min.

11. The method according to claim 2, characterized in that The centrifugation time is 5 min.

12. The method according to claim 2, characterized in that: The centrifugal speed is 5000 rpm / min.

13. The method according to claim 2, characterized in that The preparation steps of the test solution include: the Polygonum villosa is the rhizome of Polygonum villosa, accurately weighing 0.25 g of the Polygonum villosa rhizome powder that has passed through a No. 3 sieve, ultrasonically or vortex-extracting it in 7.5 mL of 50% methanol for 5 minutes, taking it out and cooling it, weighing it, making up the lost weight with 50% methanol, shaking it evenly, centrifuging it at 5000 rpm / min for 5 minutes, taking the supernatant and filtering it through a 0.22 μm organic filter membrane, taking the filtrate to obtain the test solution.

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