Method for identifying different types of calculus bovis

Abstract

The invention provides a method for identifying different types of calculus bovis, and belongs to the technical field of medicines. The method comprises the following steps: S1, extracting calculus bovis: extracting a sample by using a deuterated reagent to obtain a deuterated reagent extract of calculus bovis; s2, performing NMR detection on the deuterated reagent extract of the calculus bovis obtained in the step S1 to obtain a 1H NMR spectrum and a COSY spectrum; and S3, determining the type of the calculus bovis based on the 1H NMR spectrum and the COSY spectrum obtained in the step S2. According to the method disclosed by the invention, the bilirubin component in the calculus bovis is extracted by directly adopting the deuterated reagent, so that complex pretreatment steps required by a traditional method such as HPLC (High Performance Liquid Chromatography) are omitted, meanwhile, detection can be performed without converting combined bilirubin into free bilirubin, and the detection time can be greatly shortened.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine and relates to a method for identifying different types of bezoars, and particularly relates to a method for identifying bezoar types through NMR spectra. Background Art

[0002] Calculus bovis, also known as rhinoceros gallstones and ugly treasure, is a precious traditional Chinese medicinal herb. It is derived from the dried gallstones of the bovine species Bostaurus domesticus Gmelin. The Pharmacopoeia of the People's Republic of China (2020 edition) states that bezoar is cool in nature, sweet in flavor, and enters the heart and liver meridians. Its primary effects include clearing the heart, dispelling phlegm, invigorating the orifices, cooling the liver, calming wind, and detoxifying. It has a wide range of uses and high application value.

[0003] Natural bezoar is a naturally occurring gallstone found in the gallbladder, bile duct, or hepatic duct of bovine animals (such as cattle and buffalo). It is removed from the cattle after slaughter and then dried in the shade. It is typically oval or square in shape, yellow-red to brownish-yellow in color, and has a crisp texture. Its formation is extremely rare. Natural bezoar alone cannot meet medical and pharmaceutical needs, so bezoar substitutes have emerged. Currently approved substitutes include artificial bezoar (ACB), cultured bezoar (CCB), and in vitro cultured bezoar (CBS).

[0004] Artificial bezoar is derived from ox bile powder, bile acid, hyodeoxycholic acid, taurine, bilirubin, cholesterol, and trace elements. Due to its simple production process, it can be industrialized and widely used in clinical practice. Artificial bezoar is inexpensive and commands a significant share of the bezoar market. It is widely used in the preparation of non-emergency medications such as Niuhuang Jiedu Tablets.

[0005] In vitro cultivation of bezoar is achieved by simulating the process of bezoar formation in the cow's body. It uses fresh bile from the cow as the mother liquor, adds deoxycholic acid, bile acid and complex bilirubin calcium, and is made in a machine. Its properties, structure and chemical composition are similar to those of natural bezoar. It has a short production cycle, controllable quality, and is suitable for industrial production. It is the main substitute for bezoar.

[0006] The prices of different types of bezoar vary greatly, resulting in a large number of inferior products or dyed products being sold as fake products on the market. Therefore, it is necessary to use various analytical methods to identify the type and evaluate the quality of bezoar. The main components of bezoar are relatively complex, one of which is bilirubin, whose structure is as follows: .

[0007] Bilirubin exists in bezoar primarily in two forms: free bilirubin and conjugated bilirubin. Artificial bezoar contains primarily free bilirubin, while natural and in vitro cultured bezoar contain only a small amount of free bilirubin, with the majority being conjugated bilirubin. Currently, identification of bilirubin in bezoar is primarily achieved through chromatographic methods, such as high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC). These methods are complex, require tedious pretreatment, and cannot distinguish between conjugated and free bilirubin.

[0008] For example, Chinese patent application publication number CN117907487A discloses a method for identifying natural bezoar and its substitutes. This method uses ultra-high performance liquid chromatography-hybrid quadrupole orbital mass spectrometry to qualitatively characterize the bile acid components of natural bezoar and its substitutes. The bile acid components used include cholic acid, taurodeoxycholic acid, taurolithocholic acid, chenodeoxycholic acid, hyodeoxycholic acid, and deoxycholic acid, and a function is established for analysis. This method requires multiple reference substances and is complex, resulting in significant limitations.

[0009] The Chinese patent application with publication number CN115078440A discloses a method for 1 H NMR is used to identify the quality of bezoar. This method also identifies the type and quality of bezoar by detecting components such as taurocholic acid, glycocholic acid, hyodeoxycholic acid, taurine, and choline. However, the method for identifying the type of bezoar is relatively complex.

[0010] Therefore, there is an urgent need for a simple, efficient and fast method to identify different types of bezoar and better control the quality of bezoar medicinal materials. Summary of the Invention

[0011] In view of this, in view of the problems existing in the prior art, the object of the present invention is to provide a method for distinguishing different types of bezoars. The method can distinguish different types of bezoars by detecting different states of a single substance, the method is simple, and the detection time is shortened.

[0012] To achieve the above object of the invention, the present invention provides a method for identifying different types of bezoar, comprising the following steps: S1. Extracting bezoar: extracting the sample using a deuterated reagent to obtain a deuterated reagent extract of bezoar; S2, the deuterated reagent extract of the bezoar obtained in step S1 is subjected to NMR detection, and the obtained 1 H NMR spectrum and COSY spectrum; S3. Determine the type of bezoar based on the 1H NMR spectrum and COSY spectrum obtained in step S2.

[0013] Furthermore, in step S1, the bezoar includes natural bezoar, artificial bezoar and in vitro cultured bezoar.

[0014] Furthermore, in step S1, the deuterated reagent is selected from at least one of deuterated chloroform and deuterated DMSO.

[0015] Furthermore, in step S1, the deuterated reagent is deuterated chloroform or deuterated DMSO.

[0016] Furthermore, the extraction of bezoar in step S1 is specifically as follows: mixing bezoar with a deuterated reagent, ultrasonicating for 20-40 minutes, filtering or centrifuging, and taking the supernatant.

[0017] Furthermore, the centrifugal speed is 10000 rpm and the centrifugal time is 10 min.

[0018] Furthermore, in step S1, the weight-to-volume ratio of bezoar to deuterated reagent is 0.2-0.6:9, and the unit is mg / μL.

[0019] Furthermore, in step S1, the weight-to-volume ratio of bezoar to deuterated reagent is 0.4:9, and the unit is mg / μL.

[0020] Furthermore, in step S2, the acquisition parameters of the NMR spectrum are: 16-64 scans, temperature 20-25°C, delay time 2-5s, and acquisition time 2-5s.

[0021] Furthermore, in step S2, the acquisition parameters of the NMR spectrum are: 32 scans, temperature 23°C, delay time 3s, and acquisition time 3.1024s.

[0022] Furthermore, in step S2, the acquisition parameters of the COSY spectrum are: 2-8 scans, temperature 20-30°C, receiving gain 60-70, and delay time 1-3s.

[0023] Furthermore, in step S2, the acquisition parameters of the COSY spectrum are: 4 scans, temperature 25°C, receiving gain 66, and delay time 1.5s.

[0024] Further, in step S3, in chloroform 1 Compare the peaks at 9.29 ppm, 9.27 ppm, 10.80 ppm and 10.69 ppm in the H NMR spectrum; 1 Compare the peak at 2.42 ppm in the H NMR spectrum; and compare the coupling-related peaks at 2.42 ppm with those at 1.92 ppm or 1.87 ppm in the COSY spectrum of DMSO.

[0025] On the other hand, the present invention provides an application of the above method for identifying different types of bezoar in identifying bezoar types.

[0026] Compared with the prior art, the present invention has the following beneficial effects: 1. The method of the present invention directly uses a deuterated reagent to extract the bilirubin component in bezoar, eliminating the complex pre-treatment steps required by traditional methods such as HPLC. At the same time, it can be detected without converting conjugated bilirubin into free bilirubin, and the detection time can be greatly shortened.

[0027] 2. The method of the present invention effectively eliminates false positive results caused by adulteration with pigments or other substances through dual-solvent cross-validation combined with nuclear magnetic resonance (NMR) spectroscopy signals. It also overcomes the limitations of single-solvent testing and significantly improves identification accuracy and reliability.

[0028] 3. In the method of the present invention, the NMR signals of other components in bezoar do not overlap with the characteristic signal of bilirubin, and can be directly analyzed by NMR spectrum qualitative analysis without additional purification, with good reproducibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is the free bilirubin dissolved in different deuterated reagents 1 H NMR detection spectrum.

[0030] Figure 2 It is a mixture of cultured bezoar, natural bezoar, artificial bezoar extract and free bilirubin in deuterated chloroform. 1 H NMR spectrum.

[0031] Figure 3 It is a mixture of cultured bezoar, natural bezoar, artificial bezoar extract and free bilirubin in deuterated DMSO. 1 H NMR spectrum.

[0032] Figure 4 This is the COSY spectrum of free bilirubin in deuterated DMSO.

[0033] Figure 5 This is the COSY spectrum of artificial bezoar extract in deuterated DMSO.

[0034] Figure 6 This is the COSY spectrum of the in vivo cultured bezoar extract in deuterated DMSO.

[0035] Figure 7 This is the COSY spectrum of natural bezoar extract in deuterated DMSO.

[0036] Figure 8 The extracts of cultured bezoar, natural bezoar and artificial bezoar in deuterated DMSO in Example 5 are 1 H NMR spectrum.

[0037] Figure 9This is the COSY spectrum of the artificial bezoar extract in deuterated DMSO of Example 5.

[0038] Figure 10 This is the COSY spectrum of the in vivo cultured bezoar extract in deuterated DMSO of Example 5.

[0039] Figure 11 This is the COSY spectrum of the natural bezoar extract in deuterated DMSO of Example 5.

[0040] Figure 12 The extracts of cultured bezoar, natural bezoar and artificial bezoar in deuterated DMSO in Example 6 are 1 H NMR spectrum.

[0041] Figure 13 This is the COSY spectrum of the artificial bezoar extract in deuterated DMSO of Example 6.

[0042] Figure 14 This is the COSY spectrum of the in vivo cultured bezoar extract in deuterated DMSO of Example 6.

[0043] Figure 15 This is the COSY spectrum of the natural bezoar extract in deuterated DMSO of Example 6.

[0044] Figure 16 The extracts of cultured bezoar, natural bezoar and artificial bezoar in deuterated DMSO in Example 1 are 1 H NMR spectrum.

[0045] Figure 17 This is the COSY spectrum of the artificial bezoar extract in deuterated DMSO in Comparative Example 1.

[0046] Figure 18 This is the COSY spectrum of the in vivo cultured bezoar extract in deuterated DMSO of Comparative Example 1.

[0047] Figure 19 This is the COSY spectrum of the natural bezoar extract in deuterated DMSO in Comparative Example 1.

[0048] Figure 20 The extracts of cultured bezoar, natural bezoar and artificial bezoar in deuterated methanol in Example 2 are 1 H NMR spectrum. DETAILED DESCRIPTION

[0049] Terms and Claims of the Present Invention: 1. The articles "a", "an" and "the" include plural referents unless expressly limited to one or more referents otherwise.

[0050] 2. Numerical ranges: Unless expressly stated otherwise, all ranges or ratios disclosed herein are to be understood to include any and all subranges or subratios contained therein. For example, a range or ratio stated as 1 to 30 is to be considered inclusive of any and all subranges or subratios, integers, decimals, or subranges or subratios comprised therein, between a minimum of 1 and a maximum of 30, including any subranges or subratios, integers, decimals, or subranges or subratios comprised therein.

[0051] The following non-limiting examples are provided to enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way. The following are merely illustrative of the scope of the present invention, and those skilled in the art may make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present invention.

[0052] The present invention will be further described below by way of specific examples. The various chemical reagents used in the examples of the present invention were obtained through conventional commercial channels unless otherwise specified. Unless otherwise specified, the contents described below are all by weight. Unless otherwise specified, it is understood that the experiments were conducted at room temperature.

[0053] In the following examples, the sources of some reagents are as shown in Table 1: Table 1

[0054] Example 1 Selection of solvent for extraction of free bilirubin Take four 2 mg portions of free bilirubin powder and place them in centrifuge tubes. Add 600 μL of deuterated water, deuterated methanol, deuterated chloroform, or deuterated DMSO to each tube and vortex to mix thoroughly. Centrifuge (10,000 rpm, 10 min) or filter through a membrane to remove the supernatant and transfer it to an NMR tube.

[0055] The above four solutions were 1 H NMR detection, scan 32 times, temperature 23 ° C, delay time 3s, acquisition time 3.1024s, obtained as follows Figure 1 shown 1 H NMR spectrum.

[0056] from Figure 1 As can be seen, free bilirubin shows no signal in deuterated water and deuterated methanol, demonstrating its poor solubility in these two solutions. However, in deuterated chloroform and deuterated DMSO, the NMR signal of free bilirubin is clearly visible, with a flat baseline. Therefore, deuterated chloroform and deuterated DMSO were selected as extraction solvents and NMR reagents for bilirubin-like substances in the following examples.

[0057] Figure 1As shown in the figure, in deuterated chloroform, the singlets at 10.80ppm (s, 1H) and 10.69ppm (s, 1H) are the signals of 21-H and 24-H on the bilirubin amide bond, and the singlets at 9.29ppm (s, 1H) and 9.27ppm (s, 1H) are the signals of 23-H and 22-H on the bilirubin amino group. In deuterated DMSO: 11.89ppm (s, 2H) is the signal of 8c-H and 12c-H on the bilirubin carboxyl group, 10.48ppm (s, 1H) and 10.45ppm (s, 1H) are the signals of 23-H and 22-H on the bilirubin amino group, and 10.04ppm (s, 1H) and 9.91ppm (s, 1H) are the signals of 21-H and 24-H on the bilirubin amide bond.

[0058] Example 2 The deuterated chloroform extraction of bezoar from different sources was used to analyze the 1 H NMR detection and analysis The detection process includes the following steps: S1. Extraction of Bezoar: Powder 40 mg of each of the three bezoar samples listed in Table 1 (natural bezoar TR1, in vitro cultured bezoar TP1, and artificial bezoar RG1) and place them in separate centrifuge tubes. Add 900 μL of deuterated chloroform and vortex to mix. Wrap the tubes in foil and ultrasonically extract for 30 minutes. Pass the tubes through a 0.22 μm filter membrane and place them into NMR tubes.

[0059] S2, the three solutions were 1 H NMR detection, scan 32 times, temperature 23 ° C, delay time 3s, acquisition time 3.1024s, obtained as follows Figure 2 shown 1 H NMR spectrum.

[0060] from Figure 2 It can be seen that in deuterated chloroform, the hydrogen spectrum signal of free bilirubin can be seen in both cultured bezoar and artificial bezoar, but not in natural bezoar, proving that the free bilirubin content in natural bezoar is very low.

[0061] Example 3 The deuterated DMSO extraction of bezoar from different sources was carried out. 1 H NMR and COSY detection and analysis The detection process includes the following steps: S1. Bezoar Extraction: Powder 40 mg of each of the three bezoar samples listed in Table 1 (natural bezoar TR1, in vitro cultured bezoar TP1, and artificial bezoar RG1) and place them in separate centrifuge tubes. Add 900 μL of deuterated DMSO and vortex to mix. Wrap the tubes in foil and ultrasonically extract for 30 minutes. Centrifuge (10,000 rpm, 10 minutes) and collect the supernatant into an NMR tube.

[0062] S2, the three solutions were 1 H NMR detection, scan 32 times, temperature 23 ° C, delay time 3s, acquisition time 3.1024s, obtained as follows Figure 3 shown 1 H NMR spectrum.

[0063] S3. The three solutions and the solution of bilirubin in DMSO were subjected to COSY NMR detection, scanned 4 times, at a temperature of 25°C, a receiving gain of 66, and a delay time of 1.5s, and the following was obtained: Figure 4-Figure 7 COSY spectrum shown.

[0064] like Figure 3 、 Figure 4-Figure 7 As shown, in deuterated DMSO, the -CH2- hydrogen signals of free bilirubin connected to the carboxyl group, namely 8b-H and 12b-H, appear at 1.92 ppm, and HH coupling related peaks can be seen on the COSY spectrum with the peaks of 8a-H and 12a-H at 2.42 ppm (t, J = 9.71 Hz, 4H), and the peak height is 1-1.5 times the peak height at 5.0 ppm-7.00 ppm.

[0065] In artificial bezoar, a weak signal at 2.42ppm can be seen, proving the presence of trace amounts of free bilirubin. From the COSY spectrum, it can be seen that there are 2.42ppm and 1.87ppm. 1 H- 1 H coupling may be due to the fact that the microenvironment of bilirubin in bezoar differs from that of free bilirubin, leading to a deviation in chemical shift. In natural and in vitro cultured bezoar, the peak at 2.42 ppm is invisible, possibly due to the influence of proteins. However, the peaks at 5.0 ppm-7.00 ppm are still clearly visible and have high peak intensity, and there is no relevant coupling in the COSY spectrum, indicating that the DMSO extraction of these two bezoars yields conjugated bilirubin.

[0066] At the same time, due to the influence of protein binding, peak broadening phenomenon appeared in the spectra of these two bezoars, further confirming the existence of conjugated bilirubin.

[0067] Overall, through multi-faceted comparisons using multiple solvents, it is possible to distinguish natural bezoar, in vitro cultured bezoar and artificial bezoar by differentiating the presence of bilirubin in these three samples.

[0068] Example 4 Summary of the characteristics of different types of bezoar in the spectrum Natural bezoar: No free bilirubin signal is visible in deuterated chloroform, and no signal at 2.42 ppm is visible in deuterated DMSO, with peak broadening.

[0069] In vitro culture of bezoar: free bilirubin signal is visible in deuterated chloroform, but the signal at 2.42ppm in deuterated DMSO is not visible, and there is peak broadening.

[0070] Artificial bezoar: Free bilirubin signal is visible in deuterated chloroform, and a signal at 2.42 ppm is visible in deuterated DMSO. HH coupling correlation peaks are visible at 2.42 ppm and 1.87 ppm in the COSY spectrum.

[0071] Example 5 Follow the steps below to test the bezoar sample: S1. Extraction of Bezoar: Take 20 mg of each of the three bezoar samples listed in Table 1 (TR2, TP2, and RG1), place them in separate centrifuge tubes, add 900 μL of deuterated DMSO to each tube, and vortex to mix thoroughly. Wrap the tubes in foil and ultrasonically extract for 20 minutes. After centrifugation (10,000 rpm for 10 minutes), collect the supernatant and transfer it to an NMR tube.

[0072] S2, the above solutions are respectively 1 H NMR detection, scan 16 times, temperature 20 ° C, delay time 2s, acquisition time 2s, the following Figure 8 shown 1 H NMR spectrum.

[0073] S3. Each deuterated DMSO solution was subjected to COSY NMR detection, scanned 8 times, at a temperature of 20°C, a receiving gain of 60, and a delay time of 1s, and the following was obtained: Figures 9-11 COSY spectrum shown.

[0074] from Figures 8-11 As can be seen in the figure, in artificial bezoar, a weak signal at 2.42ppm is visible on the hydrogen spectrum, and there is a related coupling on the COSY spectrum, which proves the presence of free bilirubin. In natural bezoar and in vitro cultured bezoar, the peak at 2.42ppm is not visible, while the peak at 5.0ppm-7.00ppm is still clearly visible with high peak intensity. At the same time, there is no related coupling on the COSY spectrum, indicating that the DMSO extraction of these two bezoars is conjugated bilirubin, and both of them have the phenomenon of peak broadening.

[0075] Example 6 Follow the steps below to test the bezoar sample: S1. Extraction of Bezoar: Take 60 mg of each of the three bezoar samples listed in Table 1 (TR2, TP2, and RG1), place them in separate centrifuge tubes, add 900 μL of deuterated DMSO to each tube, and vortex to mix thoroughly. Wrap the tubes in foil and ultrasonically extract for 40 minutes. After centrifugation (10,000 rpm for 10 minutes), collect the supernatant and transfer it to an NMR tube.

[0076] S2, the above solutions are respectively 1 H NMR detection, scan 64 times, temperature 25 ° C, delay time 5s, acquisition time 5s, the following Figure 12 shown 1 H NMR spectrum.

[0077] S3. Each deuterated DMSO solution was subjected to COSY NMR detection, scanned twice, at a temperature of 30°C, a receiving gain of 70, and a delay time of 3s, to obtain the following: Figure 13-15 COSY spectrum shown.

[0078] from Figure 12-15 As can be seen in the figure, in artificial bezoar, a weak signal at 2.42ppm is visible on the hydrogen spectrum, and there is a related coupling on the COSY spectrum, which proves the presence of free bilirubin. In natural bezoar and in vitro cultured bezoar, the peak at 2.42ppm is not visible, while the peak at 5.0ppm-7.00ppm is still clearly visible with high peak intensity. At the same time, there is no related coupling on the COSY spectrum, indicating that the DMSO extraction of these two bezoars is conjugated bilirubin, and both of them have the phenomenon of peak broadening.

[0079] Comparative Example 1 The difference from Example 5 is that the temperature in steps S2 and S3 is set to 40°C, and the remaining steps are the same as Example 5. Figure 16-Figure 19 shown 1 H NMR spectrum and COSY spectrum.

[0080] from Figure 16-Figure 19 It can be seen that when the detection temperature is 40℃, the resolution of the hydrogen spectra and COSY spectra of the three bezoars measured is reduced. In the hydrogen spectrum, the signal of artificial bezoar at 2.42ppm is not visible, and the peak signal at 5.0ppm-7.00ppm is basically invisible, and the spectrum recognition effect is poor; in the COSY spectrum, the HH coupling correlation peaks of artificial bezoar at 2.42ppm and 1.87ppm are also invisible, and the three bezoars cannot be distinguished.

[0081] Comparative Example 2 The difference from Example 5 is that the extraction solvent deuterated DMSO is replaced by deuterated methanol, and the remaining steps are the same as Example 5. Figure 20 shown 1 H NMR spectrum. In Example 1, it was demonstrated that free bilirubin has poor solubility in deuterated methanol. Figure 20 There is no bilirubin signal on the hydrogen spectrum shown, and the three types of bezoar cannot be distinguished by the bilirubin signal.

[0082] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for identifying different types of bezoars, characterized in that: The following steps are involved: S1. Extracting bezoar: extracting the sample using a deuterated reagent to obtain a deuterated reagent extract of bezoar; S2, the deuterated reagent extract of the bezoar obtained in step S1 is subjected to NMR detection, and the obtained 1 H NMR spectrum and COSY spectrum; S3, based on step S2 1 The species of bezoar was confirmed by H NMR and COSY spectra.

2. The method according to claim 1, characterized in that In step S1, the bezoar includes natural bezoar, artificial bezoar and in vitro cultured bezoar.

3. The method according to claim 1, characterized in that In step S1, the deuterated reagent is selected from at least one of deuterated chloroform and deuterated DMSO.

4. The method according to claim 1, wherein In step S1, the deuterated reagent is deuterated chloroform or deuterated DMSO.

5. The method according to claim 1, wherein The step S1 of extracting bezoar is specifically as follows: mixing bezoar with a deuterated reagent, ultrasonicating for 20-40 minutes, filtering or centrifuging, and taking the supernatant.

6. The method according to claim 5, characterized in that The centrifugal speed is 10000 rpm, and the centrifugal time is 10 min.

7. The method according to claim 1, characterized in that In step S1, the weight-to-volume ratio of bezoar to deuterated reagent is 0.2-0.6:9, and the unit is mg / μL.

8. The method according to claim 1, characterized in that In step S2, the acquisition parameters of the NMR spectrum are: 16-64 scans, temperature 20-25°C, delay time 2-5s, and acquisition time 2-5s.

9. The method according to claim 1, characterized in that In step S2, the acquisition parameters of the COSY spectrum are: 2-8 scans, temperature 20-30°C, receiving gain 60-70, and delay time 1-3s.

10. Use of the method according to any one of claims 1 to 9 in identifying bezoar species.

Images