A method for establishing fingerprint spectrum of Uyghur medicine Hyssopus officinalis

Abstract

The present invention relates to a method for establishing a fingerprint of Hyssopus officinalis, and specifically to a method for determining the fingerprint of 18 characteristic peaks of Hyssopus officinalis, including neochlorogenic acid, chlorogenic acid, diosmin, rosmarinic acid, salvianolic acid B, and montanin, by using an HPLC method. In the method for establishing the fingerprint of the present invention, 328 nm is selected as the measurement wavelength; acetonitrile-0.1% phosphoric acid is used as the mobile phase and gradient elution is performed; preferably 70% methanol is used as the extraction solvent and the extraction method is heated under reflux for 40 minutes. The established method can accurately reflect the overall chemical characteristics of Hyssopus officinalis, is simple and quick, and provides a scientific basis for comprehensively improving its quality.

Description

Technical Field

[0001] The invention belongs to the technical field of ethnic medicine, and particularly relates to a method for establishing a fingerprint spectrum of the Uyghur medicine Hyssopus officinalis. Background of the Invention

[0002] Hyssopus cuspidatus Boiss., the dried aerial part of the Lamiaceae family Hyssopus genus, is a perennial herb or subshrub primarily grown in the Altay region of northern Xinjiang. It is a common medicinal herb in Uyghur medicine, commonly known as Hyssopus cuspidatus. Its Uyghur name, "Zufa," is dry and hot in nature, with a strong fragrance. It is known for its cough-relieving, expectorant, asthma-relieving, and lung-tonifying properties, making it a common treatment for coughs, colds, and asthma. Modern pharmacological research has shown that Hyssopus cuspidatus has multiple pharmacological effects, including anti-inflammatory, antioxidant, blood sugar-lowering, and antibacterial properties. It primarily treats coughs and asthma through its anti-inflammatory and antibacterial properties.

[0003] According to modern monographs on traditional ethnomedicine, both domestic and international, the origin of "Zufa" is from plants of the genera Schizonepeta and Hyssopus, primarily using their aerial parts. References primarily cite Hysspous officinais L., Hyssopus cuspidatus Boiss., and Nepeta bracteata Benth. as authentic, though customary substitutes and substitutes exist in different regions. In Chinese literature such as Xinjiang Traditional Chinese Medicine, Volume 1 of the Uyghur Pharmacopoeia, and the Flora of Xinjiang, the origin of "Zufa" is Hyssopus cuspidatus Boiss. Initially, Hyssopus cuspidatus Boiss. was mostly wild. However, in recent years, resource shortages have limited market supply, leading to widespread cultivation of Hyssopus cuspidatus Boiss. in Xinjiang. Investigations indicate that its introduction primarily stems from two sources: local varieties and imported varieties. The current quality standard "Specifications for the Preparation of Xinjiang Traditional Chinese Medicine and Uyghur Medicinal Pieces" (2010 edition) only includes items such as properties, microscopic identification, and inspection. The standards are relatively simple and cannot effectively control product quality. In view of the existence of some of the above problems, and in order to better evaluate the quality of this variety, we have carried out relevant research on it. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for analyzing the fingerprint of Hyssopus officinalis. A fingerprint determination method of 18 characteristic peaks including neochlorogenic acid, chlorogenic acid, diosmin, rosmarinic acid, salvianolic acid B, and montanin is established by HPLC.

[0005] To solve the above problems, the present invention adopts the following technical solution: a method for establishing a fingerprint spectrum of the Uyghur medicine Hyssopus officinalis, comprising the following steps:

[0006] a. Prepare mixed reference solution;

[0007] b. Prepare the test solution;

[0008] c. Determination method: Accurately pipette 10 μl of the mixed reference solution and test solution respectively, inject into liquid chromatograph, and determine the result;

[0009] d. Using the Chinese medicine chromatographic fingerprint similarity evaluation system software, with S1 as the reference spectrum, the median as the benchmark, and the time window width set to 0.1, the fingerprint spectrum was generated through multi-point correction and full spectrum peak matching;

[0010] Chromatographic conditions:

[0011] Chromatographic column: Boston Green ODS 4.6×250mm, 5μm,

[0012] Column temperature: 30°C,

[0013] Flow rate: 1ml / min,

[0014] Detection wavelength: 328nm,

[0015] Injection volume: 10 μl,

[0016] Acetonitrile was used as mobile phase A, 0.1% phosphoric acid aqueous solution was used as mobile phase B, and the gradient elution program was shown in the table below.

[0017] Mobile phase gradient elution program

[0018]

[0019] Preferably, in step a, the preparation of the mixed reference solution is as follows: the reference substances of neochlorogenic acid, chlorogenic acid, diosmin, rosmarinic acid, salvianolic acid B and montmorillonate are weighed, and 70% methanol is added to prepare a mixed reference solution containing 1 μg, 3 μg, 10 μg, 8 μg, 2 μg and 3 μg of the above reference substances per 1 ml, respectively.

[0020] Preferably, in the step b, the test solution; after the product powder is passed through a No. 3 sieve, 0.3 g is taken, weighed, placed in a stoppered conical flask, 100 ml of 70% methanol is added, the bottle is sealed, the weight is weighed, heated under reflux for 40 minutes, cooled, and the weight is weighed again, and the lost weight is supplemented with 70% methanol, shaken, filtered, and the filtrate is obtained.

[0021] Preferably, in step c, 10 μl of each of the mixed reference solution and the test solution are accurately aspirated, and 10 μl of each is injected for analysis according to the chromatographic conditions, and the chromatogram is recorded.

[0022] Beneficial effects:

[0023] The fingerprint method of the present invention uses a measurement wavelength of 328 nm, a mobile phase of acetonitrile and 0.1% phosphoric acid, and a gradient elution method. The extraction solvent is preferably 70% methanol, and the extraction is performed under heating and reflux for 40 minutes. The established method accurately reflects the overall chemical characteristics of Hyssopus sclerotium wilfordii. It is simple and rapid, providing a scientific basis for comprehensively improving its quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the HPLC chromatogram of Hyssopus officinalis mixed reference sample;

[0025] Figure 2 This is the HPLC chromatogram of Hyssopus officinalis sample;

[0026] Figure 3 This is the durability chart of Boston Green ODS (4.6×250mm, 5μm) chromatographic column;

[0027] Figure 4 This is the durability chart of COSMOSIL C18 (4.6×250mm, 5μm) chromatographic column;

[0028] Figure 5 This is the durability graph of the waters C18 (4.6×250mm, 5μm) chromatographic column;

[0029] Figure 6 This is a diagram of the durability of the Waters e2695 instrument;

[0030] Figure 7 This is the durability chart of the Shimadzu LC-20AT instrument;

[0031] Figure 8 This is the Ultimate3000 instrument durability map;

[0032] Figure 9 Superimpose fingerprints and control fingerprints for all 14 batches of Hyssopus officinalis samples;

[0033] Figure 10 Superimpose fingerprints and reference fingerprints for 10 batches of local Hyssopus officinalis samples;

[0034] in: Figure 1 Middle peak 3, neochlorogenic acid; Peak 9, chlorogenic acid peak; Peak 14, diosmin; Peak 15, rosmarinic acid; Peak 16, salvianolic acid B; Peak 18, montanin. DETAILED DESCRIPTION

[0035] The present invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the relevant invention, rather than to limit the invention.

[0036] Example

[0037] 1. Instruments and test drugs

[0038] 1.1 Instrument

[0039] High performance liquid chromatography (equipped with quaternary pump, DAD detector, Waters e2695); analytical balance: MettlerXPE26 (1 part per million) (Shanghai Mettler Instrument Co., Ltd.); Mettler XS105 (1 part per hundred thousand) (Shanghai Mettler Instrument Co., Ltd.); ultrapure water instrument (Millipore, USA).

[0040] 1.2 Drug testing

[0041] Chlorogenic acid reference substance (batch number: 110753-202119, content calculated as 96.3%);

[0042] Rosmarinic acid reference substance (batch number: 111871-202007, content calculated as 98.1%);

[0043] Salvianolic acid B reference substance (batch number: 111562-201917, content calculated as 96.6%);

[0044] Limonene reference substance (batch number: 111528-202112, content calculated as 98.0%);

[0045] The above four reference substances were purchased from China Food and Drug Inspection Institute.

[0046] The new chlorogenic acid reference substance (batch number: DSTDX001504, content ≥98%) was purchased from Lemeitian Pharmaceutical Dester Biotechnology Co., Ltd.

[0047] Diosmin reference substance (batch number: 0518-RD-0011, content 94.5%) was purchased from Guangzhou Jiatu Technology Co., Ltd.

[0048] 14 batches of Hyssopus officinalis samples: all provided by the Xinjiang Uygur Autonomous Region Institute of Drug Control. Detailed information is shown in Table 1.

[0049] Table 1 Information of 14 batches of Hyssopus officinalis samples

[0050]

[0051] Reagents: acetonitrile (chromatographic grade, Merck, Germany), water was ultrapure water; other reagents were of analytical grade.

[0052] 2. Methods and Results

[0053] 2.1 Chromatographic conditions

[0054] Chromatographic column: Boston Green ODS (4.6×250 mm, 5 μm); acetonitrile as mobile phase A, 0.1% aqueous phosphoric acid as mobile phase B, gradient elution program as shown in Table 2; column temperature: 30°C, flow rate: 1 ml / min; detection wavelength: 328 nm; injection volume: 10 μl.

[0055] Table 2 Mobile phase gradient elution program

[0056]

[0057] 2.2 Solution preparation

[0058] 2.2.1 Preparation of mixed reference solution

[0059] Accurately weigh 3.125 mg of neochlorogenic acid, 5.879 mg of chlorogenic acid, 20.577 mg of diosmin, 16.021 mg of rosmarinic acid, 4.127 mg of salvianolic acid B, and 6.057 mg of montanaside, and place them in a 200 mL volumetric flask respectively. Add an appropriate amount of 70% methanol and sonicate to dissolve them. Cool to room temperature, dilute to the mark with 70% methanol, accurately measure 10 mL of each of the above solutions and place them in the same 100 mL volumetric flask, dilute with 70% methanol and dilute to the mark to obtain the mixed reference solution.

[0060] 2.2.2 Preparation of test solution

[0061] Take the sample powder numbered SXC-9 (passed through No. 3 sieve), accurately weigh 0.3015 g, place it in a stoppered conical flask, accurately add 100 ml of 70% methanol, stopper it tightly, weigh it, heat it under reflux in a water bath at 85°C for 40 minutes, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0062] 2.3 Assay

[0063] Accurately pipette 10 μl of each mixed reference solution and test solution, and analyze them according to the chromatographic conditions under "2.1". Record the chromatogram ( Figure 1 、 Figure 2 In the sample chromatogram, the chromatographic peaks with the same retention time as those of the reference substances neochlorogenic acid, chlorogenic acid, diosmin, rosmarinic acid, salvianolic acid B and montanol were shown, with good peak shapes and the separation degree meeting the requirements.

[0064] 2.4 Building a Graph

[0065] The Chinese medicine chromatographic fingerprint similarity evaluation system software was used, with S1 as the reference spectrum, the median as the benchmark, and the time window width set to 0.1. The fingerprint spectrum was generated through multi-point correction and full spectrum peak matching.

[0066] The present invention is verified by methodology as follows:

[0067] 3.1 Selection of detection wavelength

[0068] Six reference solutions were scanned at 200nm to 400nm. The maximum absorption wavelengths of the six reference solutions were 326nm, 326nm, 345nm, 328nm, 287nm, and 333nm, respectively. Testing was performed at the maximum absorption wavelengths of the six components. The results showed that the fingerprint at 328nm exhibited more chromatographic peaks with good peak shape and met the required resolution. Therefore, 328nm was selected as the measurement wavelength for this experiment.

[0069] 3.2 Selection of extraction solvent

[0070] The extraction performance of samples with methanol, 70% methanol, 30% methanol, ethanol, 70% ethanol, and 30% ethanol was investigated. The results showed that the components of Hyssopus sclerotium were best extracted in 70% methanol, so 70% methanol was selected as the extraction solvent for this experiment.

[0071] 3.3 Selection of mobile phase

[0072] The effects of different mobile phase systems (acetonitrile-water, acetonitrile-0.1% phosphoric acid, and methanol-0.1% phosphoric acid) on the chromatographic peaks were investigated. The results showed that when the mobile phase system was acetonitrile-0.1% phosphoric acid solution (gradient elution), the chromatogram had a stable baseline, good peak shape, and better separation.

[0073] 3.4 Precision test

[0074] Take hard-pointed Hyssopus officinalis powder (numbered SXC-9), prepare the sample according to the method under "2.2.2", and measure it continuously six times according to the chromatographic conditions under "2.1", and record the chromatogram. Using chlorogenic acid as the reference peak (S1), the relative retention time and relative peak area of ​​the common peaks (peaks 1-8, peaks 10-12) were calculated, and the RSD values ​​were calculated; using rosmarinic acid as the reference peak (S2), the relative retention time and relative peak area of ​​the common peaks (peaks 13, peak 14, peaks 16-18) were calculated, and the RSD values ​​were calculated; the results are shown in Tables 3 and 4. The relative retention time RSD values ​​of each characteristic peak were less than 0.3%, and the RSD values ​​of the relative peak area were less than 1.2%, indicating good instrument precision.

[0075] Table 3 Precision relative retention time results

[0076]

[0077]

[0078] Table 4 Precision relative peak area results

[0079]

[0080] 3.5 Stability test

[0081] The test solution (numbered SXC-9) under "2.2.2" was measured at 0, 2, 6, 10, 16, 22, and 26 h using the chromatographic conditions under "2.1," and the chromatograms were recorded. Using chlorogenic acid as the reference peak (S1), the relative retention times and relative peak areas of the common peaks (Peaks 1-8, Peaks 10-12) were calculated, along with the RSD values. Using rosmarinic acid as the reference peak (S2), the relative retention times and relative peak areas of the common peaks (Peaks 13, 14, and Peaks 16-18) were calculated, along with the RSD values. The results are shown in Tables 5 and 6. The RSD values ​​for the relative retention times of each characteristic peak were less than 0.3%, and the RSD values ​​for the relative peak areas were less than 2.0%, indicating that the test solution was stable for at least 26 hours.

[0082] Table 5 Stability relative retention time results

[0083]

[0084] Table 6 Stability relative peak area results

[0085]

[0086]

[0087] 3.6 Repeatability test

[0088] A sample of Hyssopus officinalis (SXC-9) from the same batch was pulverized, and three portions of 0.15g, 0.30g, and 0.45g of the powder (passed through a No. 4 sieve) were taken. The samples were accurately weighed and prepared using the method described in "2.2.2." The samples were then assayed using the chromatographic conditions described in "2.1," and the chromatograms were recorded. Using chlorogenic acid as the reference peak (S1), the relative retention times and relative peak areas of the common peaks (peaks 1-8 and 10-12) were calculated, along with the RSDs. Using rosmarinic acid as the reference peak (S2), the relative retention times and relative peak areas of the common peaks (peaks 13, 14, and 16-18) were calculated, along with the RSDs. The results are shown in Tables 7 and 8. The RSDs for the relative retention times of each characteristic peak were less than 0.3%, and the RSDs for the relative peak areas were less than 10.0%, demonstrating good reproducibility of the method.

[0089] Table 7 Repeatability relative retention time results

[0090]

[0091] The sampling amounts of Y1-Y9 are: 0.1504g, 0.1508g, 0.1509g, 0.3019g, 0.3024g,

[0092] 0.3039g, 0.4517g, 0.4515g, 0.4519g.

[0093] Table 8 Repeatability relative peak area results

[0094]

[0095]

[0096] The sampling amounts of Y1-Y9 are: 0.1504g, 0.1508g, 0.1509g, 0.3019g, 0.3024g,

[0097] 0.3039g, 0.4517g, 0.4515g, 0.4519g.

[0098] 3.7 Durability test

[0099] 3.7.1 Column durability assessment

[0100] Three different brands of chromatographic columns were used to determine the same batch of hard-tipped Hyssopus officinalis samples (No. SXC-9). The relative retention times and relative peak areas are shown in Tables 9 and 10, and the chromatograms are shown in Tables 10 and 11. Figure 3 、 Figure 4 and Figure 5The elution time of each characteristic peak in the chromatogram of the test sample was appropriate, the separation was good, the relative retention time (RSD was less than 10%) and the relative peak area (RSD was less than 10%) were basically consistent, indicating that the chromatographic column of this method had good durability.

[0101] Table 9 Column durability relative retention time results

[0102]

[0103]

[0104] Note: Chromatographic column I: Boston Green ODS (4.6×250mm, 5μm)

[0105] Ⅱ: COSMOSIL C18 (4.6×250mm, 5μm)

[0106] Ⅲ: waters C18 (4.6×250mm, 5μm)

[0107] Table 10 Column durability relative peak area results

[0108]

[0109]

[0110] Note: Chromatographic column I: Boston Green ODS (4.6×250mm, 5μm)

[0111] Ⅱ: COSMOSIL C18 (4.6×250mm, 5μm)

[0112] Ⅲ: waters C18 (4.6×250mm, 5μm)

[0113] 3.7.2 Instrument durability assessment

[0114] The same batch of hard-pointed Hyssopus officinalis samples (No. SXC-9) were determined using the same chromatographic column (Boston Green ODS (4.6×250 mm, 5 μm) using three different brands of high performance liquid chromatographs. The results of relative retention time and relative peak area are shown in Tables 11 and 12, and the chromatograms are shown in Tables 11 and 12. Figure 6 、 Figure 7 and Figure 8 The elution time of each characteristic peak in the chromatogram of the test sample was appropriate, the separation was good, the relative retention time (RSD was less than 3%) and the relative peak area (RSD was less than 10%) were basically consistent, indicating that the instrument durability of this method was good.

[0115] Table 11 Instrument durability relative retention time results

[0116]

[0117]

[0118] Table 12 Instrument durability relative peak area results

[0119]

[0120]

[0121] 4 Establishment of fingerprint

[0122] Take 14 batches of Hyssopus officinalis samples, prepare the test solution according to the method in "2.2.2", and measure according to the chromatographic conditions in "2.1" and record the chromatogram.

[0123] The software "Similarity Evaluation System of Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)" was used. With S1 as the reference spectrum and the median as the benchmark, the time window width was set to 0.1. Through multi-point calibration and full spectrum peak matching, the superimposed fingerprints of 14 batches of Hyssopus officinalis samples and the reference fingerprints were generated. Figure 9 After matching, 18 common peaks were identified. From the similarity results of 14 batches of samples (Table 13), it can be seen that the similarity of fingerprint spectra of different batches ranges from 0.411 to 0.999, and the similarity between each batch of samples and the generated control spectra is only between 0.703 and 0.977, indicating that the overall similarity is not high and does not meet the fingerprint spectra requirements.

[0124] Table 13 Similarity evaluation results of fingerprint spectra of 14 batches of Hyssopus officinalis samples

[0125]

[0126]

[0127] The software "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" was used to generate the common patterns and reference fingerprints of 10 batches of local wild and cultivated Hyssopus officinalis samples superimposed with S1 as the control and the same parameter settings. Figure 10 The similarity results for the 10 sample batches (Table 14) show that the fingerprint similarities between different batches ranged from 0.896 to 0.998. The similarity between each batch of samples and the generated control patterns was greater than 0.9, indicating a high overall similarity and meeting the fingerprint requirements. The similarity between the four batches of introduced cultivars and the generated control patterns ranged from 0.649 to 0.781, indicating a relatively low similarity, indicating that this characteristic pattern can effectively distinguish between local samples and introduced cultivars.

[0128] Table 14 Similarity evaluation results of fingerprint spectra of 10 batches of local Hyssopus officinalis samples

[0129]

[0130]

[0131] 5 Identification of chromatographic peaks

[0132] Depend on Figure 1 It can be seen that based on the consistency of retention time and UV spectrum between the test sample chromatogram and the mixed reference sample chromatogram, a total of 6 characteristic peaks were identified, among which peak 3 was neochlorogenic acid, peak 9 was chlorogenic acid, peak 14 was diosmin, peak 15 was rosmarinic acid, peak 16 was salvianolic acid B, and peak 18 was montmorillonate.

[0133] In summary, the present invention uses 14 batches of hard-pointed Hyssopus officinalis as research objects, including 4 batches of wild products, 6 batches of local introduced cultivated products, and 4 batches of foreign introduced cultivated products. High performance liquid chromatography is used to establish the fingerprint of hard-pointed Hyssopus officinalis, and the method is simple and has strong specificity. Fingerprint is an analytical method that can comprehensively reflect the quality of drugs. It has an irreplaceable effect in the establishment of the quality control method of hard-pointed Hyssopus officinalis. The fingerprint analysis method of the effective ingredient group can not only play the purpose of comprehensively controlling the quality of drugs, but also effectively distinguish samples from different sources, thereby evaluating the quality of the product. The present invention adopts HPLC method to establish the fingerprint of hard-pointed Hyssopus officinalis, which can not only reflect its overall chemical characteristics, but also effectively distinguish local samples from foreign introduced cultivated products. The method is simple and fast, and provides a scientific basis for comprehensively improving the quality standard of hard-pointed Hyssopus officinalis.

[0134] The above description is merely a preferred embodiment of the present application and an illustration of the technical principles employed. Those skilled in the art should understand that the scope of the invention herein is not limited to the technical solutions formed by the specific combination of the above-mentioned technical features, but also encompasses other technical solutions formed by any combination of the above-mentioned technical features or their equivalents without departing from the inventive concept. For example, a technical solution formed by replacing the above-mentioned features with (but not limited to) technical features having similar functions disclosed in this application.

Claims

1. A method for establishing a fingerprint of the Uyghur medicine Hyssopus officinalis, characterized by: The following steps are involved: a. Prepare mixed reference solution: weigh the reference substances of neochlorogenic acid, chlorogenic acid, diosmin, rosmarinic acid, salvianolic acid B, and montanol, and add 70% methanol to prepare a mixed reference solution containing 1 μg, 3 μg, 10 μg, 8 μg, 2 μg, and 3 μg of the above reference substances per 1 ml, respectively; b. Prepare the test solution: After the powder of the product has passed through a No. 3 sieve, take 0.3 g, weigh it, place it in a stoppered conical flask, add 100 ml of 70% methanol, stopper it tightly, weigh it, heat and reflux for 40 minutes, let it cool, weigh it again, make up the loss with 70% methanol, shake it well, filter it, and take the filtrate to obtain the product; c. Determination method: Accurately pipette 10 μl of the mixed reference solution and the test solution respectively, inject them into the liquid chromatograph, analyze according to the chromatographic conditions, record the chromatogram, and determine the result; d. Using the Chinese medicine chromatographic fingerprint similarity evaluation system software, with the fingerprint of the first batch of Hyssopus officinalis samples as the reference spectrum, the median as the benchmark, and the time window width set to 0.1, the fingerprint spectrum was generated through multi-point correction and full spectrum peak matching; Chromatographic conditions: Chromatographic column: Boston Green ODS 4.6×250mm, 5μm, Column temperature: 30°C, Flow rate: 1ml / min, Detection wavelength: 328nm, Injection volume: 10 μl, Acetonitrile was used as mobile phase A and 0.1% phosphoric acid aqueous solution was used as mobile phase B. The gradient elution procedure was as follows:

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