Construction method and application of bile acid fingerprint spectrum in bile

Abstract

The invention relates to a construction method and application of a bile acid fingerprint spectrum in bile, and relates to the technical field of traditional Chinese medicine detection. The construction method comprises the following steps: preparing a reference substance solution: weighing at least two of taurocholic acid, taurodeoxycholic acid, glycocholic acid, cholic acid and glycodeoxycholic acid reference substances, and preparing the reference substance solution; preparing a test sample solution, namely filtering bile, taking filtrate, heating in a gradient manner, drying and grinding to obtain a test sample, and preparing the test sample solution; and constructing a fingerprint spectrum: respectively injecting the reference substance solution and the test solution into a high performance liquid chromatograph, and determining under a predetermined liquid phase condition to construct the bile acid fingerprint spectrum. The construction method can be used for fully and effectively separating and identifying various bile acid components in the bile, can be used for simultaneously detecting and measuring the various bile acid components in the bile, and has important significance in the aspects of quality control, authenticity identification, basic research on pharmacodynamic substances and the like of the bile.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine detection, and in particular to a method for constructing a bile acid fingerprint spectrum in bile and its application. Background Art

[0002] Bile acids are one of the main active components of bile, and they play a key role in the digestion and absorption of fat. Bile acids are metabolized from cholesterol in the body, and their structure is based on cholanic acid as the parent nucleus. Different bile acids are formed according to the position and number of cholanic acid substituents. According to the source, bile acids are divided into primary bile acids, secondary bile acids and tertiary bile acids, in the liver. Primary bile acids are synthesized in the liver, mainly cholic acid and chenodeoxycholic acid. After bile acids enter the intestine, the products formed are secondary bile acids, mainly deoxycholic acid, lithocholic acid, etc. The products obtained after the secondary bile acids are reabsorbed by the liver and intestine are tertiary bile acids, such as ursodeoxycholic acid.

[0003] Bile itself has high value. There are abundant records of bile drugs in traditional Chinese medicine, including bezoar, pig bile, bear bile from mammals, fish bile, snake bile from reptiles, fish, and black class, involving as many as 45 kinds of animals. Records of bile drugs can be traced back to the Han Dynasty. Dog, cow, fish bile and bezoar were recorded as early as in Shennong's Herbal Classic. On this basis, the Mingyi Bielu added pig and sheep bile, chicken bile and snake bile; it can be seen that animal bile has high medical value. Bear bile, cow bile, pig bile and chicken bile are the raw materials of the corresponding animal bile powder, among which bear bile powder is also called gold in medicine, so animal bile has high commercial value. The 2020 edition of the Chinese Pharmacopoeia records a total of 18 drugs related to animal bile, among which cow bile is an important raw material for the production of in vitro cultured bezoar, and the pharmacopoeia includes the bile acid content in bezoar and in vitro cultured bezoar into the quality standards of bezoar and in vitro cultured bezoar. This shows that the detection and identification of bile acid components in bovine bile is of great significance.

[0004] Common methods for determining bile acid include ultraviolet visible spectrophotometry, infrared spectroscopy, thin layer chromatography, nuclear magnetic resonance hydrogen spectroscopy, mass spectrometry, etc. Among them, infrared spectroscopy, nuclear magnetic resonance hydrogen spectroscopy, mass spectrometry and other detection methods have been difficult to popularize due to inaccurate quantification and high cost. Ultraviolet visible spectrophotometry and thin layer chromatography have been the detection methods for bile acid components in the early days, but ultraviolet visible spectrophotometry cannot detect multiple bile acids at the same time, and thin layer chromatography has the disadvantage of being difficult to quantify. Moreover, bile, as a natural Chinese medicinal material, is different from conventional artificially prepared drugs or chemical products containing bile acid. It contains complex components and has other interfering components such as impurities. The specific components of bile acid in bile are unknown. Therefore, it is difficult to fully and effectively separate bile acid in bile, which leads to the difficulty of effectively identifying and quality monitoring the quality of bile. Summary of the invention

[0005] In view of the above problems, the present invention provides a method for constructing a bile acid fingerprint in bile, which can fully and effectively separate and identify multiple bile acid components in bile, and can simultaneously detect and measure several bile acid components in bile. The construction method is of great significance in the quality control, authenticity identification, and basic research of drug efficacy substances of bile.

[0006] In order to achieve the above object, the present invention provides a method for constructing a bile acid fingerprint in bile, comprising the following steps:

[0007] Prepare reference solution: weigh at least two of the reference substances of taurocholic acid, taurodeoxycholic acid, glycocholic acid, cholic acid, and glycodeoxycholic acid to prepare reference solution;

[0008] Prepare the test solution: filter the bile, take the filtrate, dry it at a gradient temperature, grind it to obtain the test sample, and prepare the test solution;

[0009] Constructing a fingerprint: injecting the reference solution and the test solution into a high performance liquid chromatograph respectively and measuring them under predetermined liquid phase conditions to construct a fingerprint of bile acid.

[0010] High Performance Liquid Chromatography is called High Performance Liquid Chromatography in English, abbreviated as HPLC. It has a strong separation ability and is widely used in the determination of substance content due to its advantages such as high separation efficiency, short time, high column efficiency, short detection sensitivity, and controllable mobile phase. Evaporative Light Scattering Detector is called Evaporative Light Scattering Detector in English, abbreviated as ELSD. It has the unique property that the detected object does not need to have chromophores and chromophores; the characteristic spectrum of traditional Chinese medicine is a technical means to perform qualitative and quantitative analysis of the chemical components in traditional Chinese medicines or traditional Chinese medicine preparations through modern analytical technology, and to display this information in a graphical manner. This spectrum can help researchers and quality control personnel understand the composition of traditional Chinese medicines and ensure their quality and consistency. The inventor intends to construct a bile acid fingerprint based on HPLC-ELSD, but because bile is a natural traditional Chinese medicine, its composition is complex and has other interfering components such as impurities. Therefore, the inventor proposes to filter the bile first (if the bile is frozen and stored, it is first thawed and then filtered), and remove the mucin complex protein and flocculent fat impurities in the bile through this process. The filtrate is then dried by a gradient temperature increase, which can effectively retain the effective components of bile acid in the bile and effectively dry the bile.

[0011] In one embodiment, in the step of preparing the test solution, the preparation of the test solution includes: dissolving the test sample in a solvent, ultrasonically treating, cooling, filtering, and taking the filtrate to obtain the test solution.

[0012] In one embodiment, the ultrasonic treatment has a power of 450W-550W and a duration of 25-35min.

[0013] In one embodiment, the gradient temperature increase includes: drying at 55-65°C and drying at 85-95°C.

[0014] The relatively low drying temperature and gradient temperature increase method can retain the effective components of bile acid in bile as much as possible.

[0015] In one embodiment, the gradient temperature increase includes: drying at 55-65°C for 5-7h, and drying at 85-95°C for 3-5h.

[0016] In one embodiment, the predetermined liquid phase conditions include:

[0017] Stationary phase: chromatographic column with carbon octadecylsilyl bonded silica gel as filler;

[0018] Mobile phase: acetonitrile is used as mobile phase A, trifluoroacetic acid aqueous solution with a volume percentage concentration of 0.18-0.22% is used as mobile phase B, and gradient elution is adopted.

[0019] In one embodiment, the chromatographic column is a C18 chromatographic column with a specification of 4.6 mm×250 mm and a filler particle size of 5 μm;

[0020] The gradient elution conditions are:

[0021] From 0 to 30 min, the volume fraction of mobile phase A increased from 25% to 52%; the volume fraction of mobile phase B decreased from 75% to 48%;

[0022] 30-45min, the volume fraction of mobile phase A is 52%, and the volume fraction of mobile phase B is 48%.

[0023] In one embodiment, the predetermined liquid phase conditions further include:

[0024] Flow rate: 0.8-1.2ml / min;

[0025] Column temperature: 25-35°C;

[0026] Detector: ELSD detector.

[0027] In one embodiment, the bile includes at least one of bezoar, pig bile, bear bile, fish bile, snake bile, dog bile, sheep bile, and chicken bile; the reference substance includes taurocholic acid, taurodeoxycholic acid, glycocholic acid, cholic acid, and glycodeoxycholic acid.

[0028] The present invention also provides a fingerprint spectrum of bile acid in bile obtained by the construction method.

[0029] The present invention also provides the application of the construction method and / or the bile acid fingerprint in bile in the quality monitoring, quality description, quality evaluation and / or authenticity identification of bile.

[0030] The present invention also provides a method for detecting a bile acid fingerprint in bile, comprising the following steps:

[0031] Prepare reference solution: Prepare reference solution;

[0032] Prepare the test solution: weigh the bile to be tested as the test sample and prepare the test solution;

[0033] Detection: Pipette the above-mentioned reference solution and test solution respectively, inject them into high performance liquid chromatography and measure them under predetermined liquid phase conditions;

[0034] Evaluation: Compare the chromatogram of the test solution obtained by the above test with the bile acid fingerprint in the bile, calculate the similarity, and evaluate the quality of the bile.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The invention discloses a method for constructing a bile acid fingerprint in bile and its application. The method can fully and effectively separate and identify a variety of bile acid components in bile, and can simultaneously detect and measure the bile acid components in a variety of biles. The method for constructing bile acid fingerprint in bile is of great significance in terms of bile quality control, authenticity identification, and basic research on drug efficacy substances. The method for detecting the bile acid fingerprint in bile can identify and detect the bile acid components contained in 5 types of bile within 50 minutes, and the method is simple, rapid, and accurate. It can provide a scientific basis for the quality evaluation and control of bile. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The HPLC chromatograms of bile acids of six batches of bovine bile samples in Example 1 and the control fingerprints are shown, wherein the seven curves from bottom to top are ZH20240624, 20240524-red, 20240524-green, Y-01-20240701, Y-01-20240801, ZH20240527, and the control fingerprint;

[0038] Figure 2 is the control fingerprint of the cow bile sample in Example 1;

[0039] Figure 3 The HPLC spectrum of the bovine bile to be tested in Example 2 (Batch No.: 20240523);

[0040] Figure 4 It is the HPLC spectrum of Comparative Example 1;

[0041] Figure 5 It is the HPLC spectrum of Comparative Example 2;

[0042] Figure 6 The HPLC spectrum of Comparative Example 3 is shown. DETAILED DESCRIPTION

[0043] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0045] Example 1

[0046] A method for constructing a fingerprint of bile acids in bile.

[0047] 1. Preparation of reference solution. Taurocholic acid, taurodeoxycholic acid, glycocholic acid, cholic acid, glycodeoxycholic acid

[0048] Accurately weigh appropriate amounts of taurocholic acid, taurodeoxycholic acid, glycocholic acid, cholic acid, and glycodeoxycholic acid reference substance, accurately weigh, and add methanol to make a solution containing 0.2 mg of taurocholic acid, 0.08 mg of taurodeoxycholic acid, 0.2 mg of glycocholic acid, 0.2 mg of cholic acid, and 0.08 mg of glycodeoxycholic acid per 1 ml.

[0049] 2. Preparation of test solution.

[0050] Take 500 ml of fresh cow bile (fresh cow bile is usually frozen for storage purposes), thaw it, pass it through a No. 8 sieve, and filter out the mucin complex and a small amount of flocculent fat impurities in the cow bile. Place them in glass culture dishes, place them in a 60°C oven to dry for 6 hours, take them out and cool them to room temperature, and place the samples in a mortar for preliminary grinding. After grinding, place the samples in a 90°C oven to continue drying for 4 hours, take them out, cool them to room temperature, and place them in a mortar for grinding until the samples are free of particles and completely in a uniform powder state, and use them as test samples for later use.

[0051] Accurately weigh about 100 mg of the test sample and place it in a stoppered conical flask. Accurately add 50 ml of methanol, weigh and record the initial weight. Use an ultrasonic processor (500 W, 53 kHz) for 30 minutes. After cooling, weigh again, calculate and add the amount of methanol lost due to evaporation. After shaking, filter through an appropriate filter membrane and take the filtrate as the test sample solution.

[0052] 3. Chromatographic conditions.

[0053] Chromatographic column: C18 column, size 4.6 mm × 250 mm, particle size 5 μm.

[0054] Mobile phase: acetonitrile-0.2% trifluoroacetic acid aqueous solution, gradient elution according to the table below.

[0055] Table 1 Gradient elution conditions

[0056]

[0057] Column temperature: 30℃.

[0058] Flow rate: 1.0ml / min.

[0059] Detector: Evaporative Light Scattering Detector (ELSD).

[0060] Accurately pipette 5μl, 10μl, 20μl of the reference solution and 10μl of the test solution respectively, and inject them into the HPLC system using a microinjector. Measure under the set chromatographic conditions to obtain the result.

[0061] Collection time: 48 minutes.

[0062] 4. Chemical identification of major chromatographic peaks.

[0063] By comparing the retention time with the reference substance, a total of 5 main characteristic peaks were identified, which were taurocholic acid, taurodeoxycholic acid, glycocholic acid, cholic acid, and glycodeoxycholic acid in order of retention time. The identified chromatographic peak area accounted for 84.5% of the total peak area, as shown in Table 2.

[0064] Table 2 Comparison of retention time of main chromatographic peaks of ox bile test solution and reference substance

[0065]

[0066] 5. Similarity calculation.

[0067] The bile acid fingerprints of 6 batches of bovine bile samples (HPLC chromatograms of 6 batches of bovine bile samples as shown in Figure 1 The CDF data file of the chromatographic fingerprint of Chinese medicine (as shown in Figure 2) was imported into the software "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2004A Edition" for analysis, and the similarity of the ox bile fingerprint was calculated with the generated reference spectrum as a reference. The results showed that the fingerprints of the six batches of ox bile samples were similar to the reference spectrum (as shown in Figure 2). Figure 2 The similarities of the six batches of bovine bile were all greater than 0.994, indicating that the quality of these six batches of bovine bile was relatively uniform and stable.

[0068] Table 3 Comparison of similarity of main chromatographic peaks of ox bile test solution

[0069]

[0070] Example 2

[0071] This embodiment is a method for detecting cow bile, and the steps are as follows:

[0072] 1. Preparation of sample solution to be tested.

[0073] Take the cow bile to be tested (batch number: 20240523) to prepare the test sample. For the specific method, refer to the "Preparation of test sample solution" in Example 1.

[0074] 2. Chromatographic conditions.

[0075] See the "Chromatographic conditions" item in Example 1.

[0076] 3. Determination: Accurately pipette 10 μl of the sample solution to be tested, inject it into a high performance liquid chromatograph, and perform the determination according to the chromatographic conditions of Example 1.

[0077] The results are as follows Figure 3 Comparing the characteristic spectrum of ox bile with that of the control established in Example 1, it can be seen that the ox bile of this batch has a high similarity with the characteristic spectrum of the control and is of qualified quality.

[0078] Comparative Example 1

[0079] It is basically the same as Example 1, except that:

[0080] The mobile phase of this comparative example is acetonitrile-0.2% formic acid aqueous solution, and the gradient elution is performed according to the following table:

[0081] Table 4 Gradient elution conditions

[0082]

[0083] like Figure 4 As shown, the sample peaks were not effectively separated.

[0084] Comparative Example 2

[0085] It is basically the same as Example 1, except that:

[0086] The mobile phase of this comparative example is acetonitrile-0.2% glacial acetic acid aqueous solution, and the gradient elution is performed according to the following table:

[0087] Table 5 Gradient elution conditions

[0088]

[0089] like Figure 5 As shown, the sample peaks were not effectively separated.

[0090] Comparative Example 3

[0091] It is basically the same as Example 1, except that:

[0092] The mobile phase of this comparative example is acetonitrile-0.2% trifluoroacetic acid aqueous solution, and isocratic elution is performed according to the following table:

[0093] Table 6 Isocratic elution conditions

[0094]

[0095] like Figure 6 As shown, the sample peaks were not effectively separated.

[0096] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0097] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for constructing a bile acid fingerprint in bile, characterized in that: The following steps are involved: Prepare reference solution: weigh at least two of the reference substances of taurocholic acid, taurodeoxycholic acid, glycocholic acid, cholic acid, and glycodeoxycholic acid to prepare reference solution; Prepare the test solution: filter the bile, take the filtrate, dry it at a gradient temperature, grind it to obtain the test sample, and prepare the test solution; Constructing a fingerprint: injecting the reference solution and the test solution into a high performance liquid chromatograph respectively and measuring them under predetermined liquid phase conditions to construct a fingerprint of bile acid.

2. The construction method according to claim 1, characterized in that: The gradient temperature increase includes: drying at 55-65° C. and drying at 85-95° C.

3. The construction method according to claim 2, characterized in that: The gradient temperature increase includes: drying at 55-65° C. for 5-7 hours, and drying at 85-95° C. for 3-5 hours.

4. The construction method according to claim 1, characterized in that: The predetermined liquid phase conditions include: Stationary phase: chromatographic column with carbon octadecylsilyl bonded silica gel as filler; Mobile phase: acetonitrile is used as mobile phase A, trifluoroacetic acid aqueous solution with a volume percentage concentration of 0.18-0.22% is used as mobile phase B, and gradient elution is adopted.

5. The construction method according to claim 4, characterized in that: The chromatographic column is a C18 chromatographic column with a specification of 4.6 mm × 250 mm and a filler particle size of 5 μm; The gradient elution conditions are: From 0 to 30 min, the volume fraction of mobile phase A increased from 25% to 52%; the volume fraction of mobile phase B decreased from 75% to 48%; 30-45min, the volume fraction of mobile phase A is 52%, and the volume fraction of mobile phase B is 48%.

6. The construction method according to claim 4, characterized in that: The predetermined liquid phase conditions also include: Flow rate: 0.8-1.2ml / min; Column temperature: 25-35°C; Detector: ELSD detector.

7. The construction method according to any one of claims 1 to 6, characterized in that: The bile includes at least one of bezoar, pig bile, bear bile, fish bile, snake bile, dog bile, sheep bile and chicken bile; the reference substance includes taurocholic acid, taurodeoxycholic acid, glycocholic acid, cholic acid and glycodeoxycholic acid.

8. The fingerprint of bile acid in bile obtained by the construction method according to any one of claims 1 to 7.

9. Application of the construction method according to any one of claims 1 to 7 and / or the bile acid fingerprint in bile according to claim 8 in quality monitoring, quality description, quality evaluation and / or authenticity identification of bile.

10. A method for detecting bile acid fingerprint in bile, characterized in that: The following steps are involved: Prepare a reference solution: prepare a reference solution corresponding to claim 1; Prepare the test solution: weigh the bile to be tested as the test sample, and prepare the test solution according to the method of claim 1; Detection: Pipette the above-mentioned reference solution and test solution respectively, inject them into high performance liquid chromatography and measure them under predetermined liquid phase conditions; Evaluation: Compare the chromatogram of the test solution obtained by the above detection with the bile acid fingerprint of bile in claim 8, calculate the similarity, and evaluate the quality of the bile.

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