Method for identifying semen armeniacae amarae and adulterants thereof
The identification of bitter almonds and peach kernels through monosaccharide mapping technology has solved the problem of identification difficulties in the existing technology, and achieved a high-accurate identification method to ensure the quality and clinical effect of medicinal materials.
Patent Information
- Application Number
- CN202510788727.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-13
- Publication Date
- 2025-07-22
AI Technical Summary
The prior art is difficult to accurately identify bitter almonds and peach kernels, which leads to serious adulteration and affects the quality and clinical effect of medicinal materials.
The monosaccharide mapping technology was used to identify the monosaccharide composition differences of bitter almonds and peach kernels through decolorization, defatization, polysaccharide extraction, fractionation purification and ion chromatography.
It provides a simple and intuitive identification method with high accuracy, which can effectively distinguish bitter almonds and peach kernels, ensuring the quality of medicinal materials and the clinical application effect.
Smart Images
Figure CN120352553A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of identification of traditional Chinese medicines and easily counterfeited products thereof, and in particular to an identification method of bitter almonds and their counterfeited counterparts, peach kernels. Background Art
[0002] Bitter almonds are the dried mature seeds of the wild apricot (prunus armeniaca L. var. Ansu. Maxim.) of the Rosaceae family. They have the effects of moistening the intestines and promoting bowel movements, anti-inflammatory, relieving cough and relieving asthma, and have a large market demand. Bitter almonds are a Chinese medicinal material that can be used as both medicine and food, and are widely planted in northern my country. Bitter almonds and peach kernels are similar in appearance, and it is difficult to distinguish them by appearance. There is also a certain difference in the price of bitter almonds and peach kernels. Under the inducement of economic interests, unscrupulous vendors usually adulterate and sell bitter almonds and their easily counterfeited peach kernels, resulting in the phenomenon of inferior products being sold as good ones and fake products being sold as real ones, resulting in poor clinical application effects of bitter almonds and their processed products, stewed bitter almonds. In order to solve the above problems, it is very meaningful to conduct authenticity identification research on bitter almonds and their easily counterfeited peach kernels.
[0003] At present, the identification of bitter almonds and their easily counterfeited counterparts, peach kernels, is often carried out by fingerprinting, but the fingerprinting operation is relatively complicated and the pre-processing steps are demanding, which may cause misjudgment. Shape identification requires the identification personnel to have strong professionalism. Therefore, it is imperative to establish a more accurate method to identify the two Chinese medicinal materials. Summary of the invention
[0004] The purpose of the present invention is to provide a method for identifying bitter almonds and their counterfeit peach kernels. The method is based on monosaccharide atlas technology, takes the number of monosaccharide chromatographic peaks of bitter almonds and peach kernels as the basis for identification, provides data support for the quality control of traditional Chinese medicines, and has important application value.
[0005] The technical solution adopted by the present invention to solve its technical problem is:
[0006] A method for identifying bitter almonds and their mixed products, peach kernels, comprises the following steps:
[0007] Step 1: Decolorization and degreasing of raw materials: Soak bitter almonds and peach kernels in 95% ethanol for a certain period of time, separate the solid and liquid, and dry them naturally;
[0008] Step 2, polysaccharide extraction: respectively mixing the decolorized and defatted bitter almonds and peach kernels with water in a certain proportion for condensation reflux extraction, filtering and combining the extracts, and obtaining the extracts of each sample after solid-liquid separation;
[0009] Step 3. Polysaccharide fractionation and purification: Concentrate the collected sample extracts, then add 95% ethanol to adjust the alcohol content to 30%-40%, take the supernatant after centrifugation, and then use 95% ethanol again to adjust the alcohol content to 70%-80%, collect the precipitate after centrifugation and freeze-dry it;
[0010] Step 4. Pretreatment of samples before injection: Take clean chromatographic vials, weigh appropriate amounts of polysaccharide samples respectively, add 2M TFA acid solution, heat at 110-125 °C for 1-5 h, pass nitrogen, and dry; Add 99.99% methanol for cleaning and then dry again. Repeat the methanol cleaning 3 times, add sterile water to dissolve, and transfer to the chromatographic vial for testing;
[0011] Step 5. Determination by ion chromatograph: Use an ion chromatograph to determine the samples to be tested in the chromatographic vials to obtain monosaccharide spectra;
[0012] Step 6. Comparison of monosaccharide spectra: Qualitatively analyze the Chinese medicinal materials Prunus armeniaca L. var. ansu Maxim. and Prunus persica (L.) Batsch according to the differences in monosaccharide composition in the monosaccharide spectra.
[0013] Preferably, in the said Step 1, the mass-volume ratio of Prunus armeniaca L. var. ansu Maxim. and Prunus persica (L.) Batsch to 95% ethanol solution is 1:5-12 g / mL, and the soaking time is 10-15 h.
[0014] Preferably, in the said Step 2, the mass-volume ratio of Prunus armeniaca L. var. ansu Maxim. and Prunus persica (L.) Batsch after decolorization and degreasing to water is 1:6-10 g / mL, the extraction temperature is 65-85 °C, and the extraction time is 2.5-3.5 h.
[0015] Preferably, in the said Step 3, each sample extract is concentrated to 1 / 30-40 of the original volume, 95% ethanol is added for the first time to adjust the alcohol content to 30%-40%, the first centrifugation speed is 3000-5000 r / min, and the centrifugation time is 6-12 min. Then use 95% ethanol again to adjust the alcohol content to 70%-80%, the second centrifugation speed is 3000-5000 r / min, and the centrifugation time is 6-12 min.
[0016] Preferably, in the said Step 4, the mass-volume ratio of polysaccharide to 2M TFA acid solution is 20-30:1 μg / mL, the heating temperature after mixing the polysaccharide sample and 2M TFA acid solution is 110-125 °C, the heating time is 1-5 h, and the concentration of the sample after being dissolved in sterile water is 20-30 μg / mL.
[0017] Preferably, in the fifth step, the sample injection volume is 5 - 10 μL, mobile phase A is H2O, mobile phase B is 0.1 M NaOH, mobile phase C is 0.1 M NaOH and 0.2 M NaAc, and the flow rate is 0.5 - 1.0 mL / min; the column temperature is 25 - 40 °C; the elution gradient: at 0 min, the ratio of phase A / phase B / phase C is 95 - 90:5 - 10:0, V / V; at 26 min, the ratio of phase A / phase B / phase C is 85 - 80:5 - 10:10, V / V; at 42 min, the ratio of phase A / phase B / phase C is 85 - 80:5 - 10:10, V / V; at 42.1 min, the ratio of phase A / phase B / phase C is 60 - 50:0:40 - 50, V / V; at 52 min, the ratio of phase A / phase B / phase C is 60 - 50:40 - 50:0, V / V; at 52.1 min, the ratio of phase A / phase B / phase C is 95 - 90:5 - 10:0, V / V; at 60 min, the ratio of phase A / phase B / phase C is 95 - 90:5 - 10:0, V / V.
[0018] Preferably, in the fifth step, the liquid chromatography column is Dionex TM CarboPac TM PA20, with a specification of 150 × 3.0 mm, 10 μm.
[0019] Preferably, in the sixth step, there are 5 monosaccharide chromatographic peaks in the monosaccharide ion chromatogram of bitter apricot kernels, representing fucose, rhamnose, arabinose, galactose, and glucose respectively; there are 3 monosaccharide chromatographic peaks in the monosaccharide ion chromatogram of peach kernels, representing fucose, galactose, and glucose respectively.
[0020] The beneficial effects produced by the present invention are as follows:
[0021] 1. The present invention identifies the monosaccharide composition of bitter apricot kernels and peach kernels, and qualitatively identifies bitter apricot kernels and peach kernels based on the monosaccharide composition differences between the two Chinese medicinal materials. The method is simple and intuitive, and can effectively identify bitter apricot kernels and peach kernels.
[0022] 2. The present invention uses ion chromatography to identify the monosaccharide composition of bitter apricot kernels and peach kernels, with high repeatability and accuracy, and can provide data support for the quality evaluation of bitter apricot kernels. Description of the Drawings
[0023] Figure 1 are the physical pictures of bitter apricot kernels and peach kernels;
[0024] Figure 2 is the monosaccharide composition ion chromatogram of bitter apricot kernels, peach kernels, and the mixed standard in Example 1;
[0025] Figure 3 is the monosaccharide composition ion chromatogram of bitter apricot kernels, peach kernels, and the mixed standard in Example 2;
[0026] Figure 4 Ion chromatogram of monosaccharide composition of bitter apricot kernels, peach kernels and mixed reference standards in Example 3. Detailed implementation manners
[0027] Next, in combination with the implementation manners of the present invention, the technical solutions in the implementation manners of the present invention will be clearly and completely described. Obviously, the described implementation manners are only a part of the implementation manners of the present invention, rather than all the implementation manners. Based on the implementation manners in the present invention, all other implementation manners obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.
[0028] The following main reagents and instruments are used in the following examples: Thermo ICS 5000+ ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA), purchased from Thermo Fisher Scientific Co., Ltd.; NaOH and NaAc, purchased from Sinopharm Chemical Reagent Co., Ltd., and the rest of the reagents are all chromatographically pure.
[0029] Example 1
[0030] This example provides a method for identifying bitter apricot kernels and their adulterated peach kernels. The specific steps of the method are as follows:
[0031] (1) First, accurately weigh 50 g of bitter apricot kernels and 50 g of peach kernels into two identical extraction containers respectively, add 250 mL of 95% ethanol to soak the bitter apricot kernels and peach kernels for 10 h, and after solid-liquid separation, air-dry naturally. Add 300 mL of pure water to the extraction containers containing the decolorized and defatted bitter apricot kernels and peach kernels respectively, and carry out reflux extraction at an extraction temperature of 65 °C for 2.5 h, filter and combine the extraction solutions to obtain 285 mL of each sample extraction solution. Then concentrate the collected sample extraction solutions to 9.5 mL, then add 95% ethanol to adjust the alcohol content to 30%, centrifuge at a centrifuge speed of 3000 r / min for 6 min to collect the supernatant, and then use 95% ethanol again to adjust the alcohol content to 70%, centrifuge at a centrifuge speed of 3000 r / min for 6 min to collect the precipitate and freeze-dry to obtain 10 mg of each of the two samples;
[0032] (2) Take clean chromatographic vials, weigh appropriate amounts of polysaccharide samples respectively, add 2M TFA acid solution to make the solution concentration 20 μg / mL, and heat at 110 °C for 1 h. Pass nitrogen and blow dry. Add 99.99% methanol for cleaning and then blow dry, repeat the methanol cleaning 3 times. Add sterile water to dissolve the sample to a concentration of 20 μg / mL, transfer it to a chromatographic vial for measurement. Use an ion chromatograph to measure the samples to be measured in the chromatographic vials to obtain monosaccharide spectra. The measurement parameters are that the liquid chromatographic column is Dionex TMCarboPac TM PA20 (150×3.0 mm, 10 μm); The injection volume was 5 μL. Mobile phase A (H2O), mobile phase B (0.1 M NaOH), mobile phase C (0.1 M NaOH, 0.2 M NaAc), flow rate 0.5 mL / min; Column temperature was 25 °C; Elution gradient: 0 min A phase / B phase / C phase (95:5:0, V / V), 26 min A phase / B phase / C phase (85:5:10, V / V), 42 min A phase / B phase / C phase (85:5:10, V / V), 42.1 min A phase / B phase / C phase (60:0:40, V / V), 52 min A phase / B phase / C phase (60:40:0, V / V), 52.1 min A phase / B phase / C phase (95:5:0, V / V), 60 min A phase / B phase / C phase (95:5:0, V / V). Finally, qualitative identification of traditional Chinese medicinal materials Prunus armeniaca and Prunus persica was carried out according to the differences in monosaccharide composition in the monosaccharide chromatogram. The ion chromatograms of the two samples are as Figure 2 shown.
[0033] Example 2
[0034] This example provides a method for identifying Prunus armeniaca and its adulterant Prunus persica. The specific steps of the method are as follows:
[0035] (1) First, accurately weigh 50 g of Prunus armeniaca and 50 g of Prunus persica into two identical extraction containers respectively, add 600 mL of 95% ethanol to soak Prunus armeniaca and Prunus persica for 15 h, and after solid-liquid separation, air-dry naturally. Add 500 mL of pure water to the extraction containers containing decolorized and defatted Prunus armeniaca and Prunus persica respectively, and perform reflux extraction at an extraction temperature of 85 °C for 3.5 h, filter and combine the extraction solutions to obtain 482 mL of extraction solution for each sample. Then concentrate the collected extraction solution of each sample to 12.1 mL, then add 95% ethanol to adjust the alcohol content to 40%, centrifuge at a centrifugal speed of 5000 r / min for 12 min to collect the supernatant, and then use 95% ethanol again to adjust the alcohol content to 80%, centrifuge at a centrifugal speed of 5000 r / min for 12 min to collect the precipitate and freeze-dry to obtain 13.4 mg of the two samples respectively;
[0036] (2) Take clean chromatographic vials, weigh appropriate amounts of polysaccharide samples respectively, add 2 M TFA acid solution until the solution concentration is 30 μg / mL, heat at 125 °C for 5 h. Pass nitrogen and blow dry. Add 99.99% methanol for cleaning and then blow dry again, repeat the methanol cleaning 3 times. Add sterile water to dissolve the sample to a concentration of 30 μg / mL, transfer it into the chromatographic vial for measurement. Use an ion chromatograph to measure the samples to be measured in the chromatographic vial to obtain a monosaccharide chromatogram. The measurement parameters are that the liquid chromatographic column is Dionex TMCarboPac TM PA20 (150×3.0 mm, 10 μm); The injection volume was 10 μL. Mobile phase A (H2O), mobile phase B (0.1 M NaOH), mobile phase C (0.1 M NaOH, 0.2 M NaAc), flow rate 1.0 mL / min; Column temperature was 40 °C; Elution gradient: 0 min A phase / B phase / C phase (90:10:0, V / V), 26 min A phase / B phase / C phase (80:10:10, V / V), 42 min A phase / B phase / C phase (80:10:10, V / V), 42.1 min A phase / B phase / C phase (50:0:50, V / V), 52 min A phase / B phase / C phase (50:50:0, V / V), 52.1 min A phase / B phase / C phase (90:10:0, V / V), 60 min A phase / B phase / C phase (90:10:0, V / V). Finally, the traditional Chinese medicinal materials bitter apricot kernels and peach kernels were qualitatively identified according to the differences in monosaccharide composition in the monosaccharide chromatogram. The ion chromatograms of the two samples are as Figure 3 shown.
[0037] Example 3
[0038] This example provides a method for identifying bitter apricot kernels and their adulterated products peach kernels. The specific steps of the method are as follows:
[0039] (1) First, accurately weigh 50 g of bitter apricot kernels and 50 g of peach kernels into two identical extraction containers respectively. Add 350 mL of 95% ethanol to soak the bitter apricot kernels and peach kernels for 12 h, and after solid-liquid separation, air-dry naturally. Add 350 mL of pure water to the extraction containers containing the decolorized and defatted bitter apricot kernels and peach kernels respectively, and carry out reflux extraction at an extraction temperature of 75 °C for 3 h, filter and combine the extraction solutions to obtain 338 mL of extraction solution for each sample. Then concentrate the collected extraction solution of each sample to 10 mL, then add 95% ethanol to adjust the alcohol content to 35%, centrifuge at a centrifugal speed of 4000 r / min for 9 min to collect the supernatant, and then use 95% ethanol again to adjust the alcohol content to 75%, centrifuge at a centrifugal speed of 4000 r / min for 9 min to collect the precipitate and freeze-dry to obtain 12.1 mg of the two samples respectively;
[0040] (2) Take clean chromatographic vials, weigh appropriate amounts of polysaccharide samples respectively, add 2 M TFA acid solution until the solution concentration is 25 μg / mL, heat at 121 °C for 3 h. Pass nitrogen and blow dry. Add 99.99% methanol for cleaning and then blow dry again, repeat the methanol cleaning 3 times. Add sterile water to dissolve the sample to a concentration of 25 μg / mL, transfer it into the chromatographic vial for testing. Use an ion chromatograph to measure the samples to be tested in the chromatographic vial to obtain a monosaccharide chromatogram. The measurement parameters are that the liquid chromatographic column is Dionex TMCarboPac TM PA20 (150×3.0 mm, 10 μm); The injection volume was 7 μL. Mobile phase A (H2O), mobile phase B (0.1 M NaOH), mobile phase C (0.1 M NaOH, 0.2 M NaAc), flow rate 0.8 mL / min; Column temperature was 30 °C; Elution gradient: 0 min A phase / B phase / C phase (92:8:0, V / V), 26 min A phase / B phase / C phase (83:7:10, V / V), 42 min A phase / B phase / C phase (81:9:10, V / V), 42.1 min A phase / B phase / C phase (55:0:45, V / V), 52 min A phase / B phase / C phase (55:45:0, V / V), 52.1 min A phase / B phase / C phase (92:8:0, V / V), 60 min A phase / B phase / C phase (92:8:0, V / V). Finally, the traditional Chinese medicinal materials Prunus armeniaca and Prunus persica were qualitatively identified according to the differences in monosaccharide composition in the monosaccharide chromatogram. The ion chromatograms of the two samples are as Figure 4 shown.
[0041] It can be seen from Figures 2-4 the figure that there are 5 monosaccharide chromatographic peaks in the monosaccharide ion chromatogram of Prunus armeniaca, representing fucose, rhamnose, arabinose, galactose and glucose respectively; there are 3 monosaccharide chromatographic peaks in the monosaccharide ion chromatogram of Prunus persica, representing fucose, galactose and glucose respectively. Prunus armeniaca and Prunus persica can be accurately distinguished according to the number of monosaccharide chromatographic peaks.
[0042] Based on the above data analysis results, the method provided by the present invention can compare and distinguish Prunus armeniaca and Prunus persica according to the differences in monosaccharide composition, with strong accuracy. The explicit identification index can highly guarantee the clinical application effect of Prunus armeniaca in the field of traditional Chinese medicine, and promote the healthy cycle and development of the Prunus armeniaca industry.
[0043] The above embodiments are only examples of the preferred embodiments of the present invention and do not cover all the implementation scopes of the invention. Those of ordinary skill in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined by the claims.
Claims
1. A method for identifying bitter apricot kernels and their adulterated products, peach kernels, characterized in that, It includes the following steps: Step 1, raw material decolorization and degreasing: First, soak bitter apricot kernels and peach kernels in 95% ethanol for a period of time respectively, and after solid-liquid separation, air-dry naturally; Step 2, polysaccharide extraction: Mix the decolorized and degreased bitter apricot kernels and peach kernels with water in a certain ratio respectively for condensing reflux extraction, filter and combine the extraction solutions, and obtain the extraction solutions of each sample after solid-liquid separation; Step 3, polysaccharide fractionation and purification: Concentrate the collected extraction solutions of each sample, then add 95% ethanol to adjust the alcohol content to 30%-40%, take the supernatant after centrifugation, and then use 95% ethanol again to adjust the alcohol content to 70%-80%, collect the precipitate after centrifugation and freeze-dry; Step 4, sample pretreatment before machine entry: Take clean chromatographic vials, weigh appropriate amounts of polysaccharide samples respectively, add 2M TFA acid solution, heat at 110-125°C for 1-5 h. Pass nitrogen and blow dry. Add 99.99% methanol for cleaning, and then blow dry again. Repeat the methanol cleaning 3 times. Add sterile water to dissolve and transfer it into the chromatographic vial for testing; Step 5, determination by ion chromatograph: Use an ion chromatograph to measure the samples to be tested in the chromatographic vials to obtain monosaccharide chromatograms; Step 6, monosaccharide chromatogram comparison: Identify genuine and easily counterfeited products of Chinese medicinal materials bitter apricot kernels and peach kernels according to the number of peaks in the monosaccharide chromatograms of each sample.
2. The preparation method according to claim 1, wherein In the said Step 1, the mass-volume ratio of bitter apricot kernels and peach kernels to 95% ethanol solution is 1:5-12 g / mL, and the soaking time is 10-15 h.
3. The preparation method according to claim 1, wherein In the said Step 2, the mass-volume ratio of the decolorized and degreased bitter apricot kernels and peach kernels to water is 1:6-10 g / mL, the extraction temperature is 65-85°C, and the extraction time is 2.5-3.5 h.
4. The preparation method according to claim 1, wherein In the said Step 3, each sample extraction solution is concentrated to 1 / 30-40 of the original volume, 95% ethanol is added for the first time to adjust the alcohol content to 30%-40%, the first centrifugation speed is 3000-5000 r / min, the centrifugation time is 6-12 min, and then 95% ethanol is used again to adjust the alcohol content to 70%-80%, the second centrifugation speed is 3000-5000 r / min, and the centrifugation time is 6-12 min.
5. The preparation method according to claim 1, wherein In the said Step 4, the mass-volume ratio of polysaccharide to 2M TFA acid solution is 20-30:1 μg / mL, the heating temperature after mixing the polysaccharide sample with 2M TFA acid solution is 110-125°C, the heating time is 1-5 h, and the concentration of the sample after being dissolved in sterile water is 20-30 μg / mL.
6. The preparation method according to claim 1, characterized in that, In step five, the sample injection volume is 5 - 10 μL, mobile phase A is H2O, mobile phase B is 0.1 M NaOH, mobile phase C is 0.1 M NaOH and 0.2 M NaAc, and the flow rate is 0.5 - 1.0 mL / min; the column temperature is 25 - 40 °C; the elution gradient: at 0 min, the ratio of phase A / phase B / phase C is 95 - 90:5 - 10:0, V / V; at 26 min, the ratio of phase A / phase B / phase C is 85 - 80:5 - 10:10, V / V; at 42 min, the ratio of phase A / phase B / phase C is 85 - 80:5 - 10:10, V / V; at 42.1 min, the ratio of phase A / phase B / phase C is 60 - 50:0:40 - 50, V / V; at 52 min, the ratio of phase A / phase B / phase C is 60 - 50:40 - 50:0, V / V; at 52.1 min, the ratio of phase A / phase B / phase C is 95 - 90:5 - 10:0, V / V; at 60 min, the ratio of phase A / phase B / phase C is 95 - 90:5 - 10:0, V / V.
7. The preparation method according to claim 1, characterized in that, In the fifth step, the liquid chromatography column is Dionex TM CarboPac TM PA20, with a specification of 150×3.0 mm and 10 μm.
8. The preparation method according to claim 1, wherein In step six, there are 5 obvious monosaccharide chromatographic peaks in the monosaccharide chromatogram of bitter apricot kernels, and 3 obvious monosaccharide chromatographic peaks in the monosaccharide chromatogram of peach kernels.
9. The preparation method according to claim 8, wherein The 5 monosaccharide chromatographic peaks in the monosaccharide ion chromatogram of bitter apricot kernels represent fucose, rhamnose, arabinose, galactose and glucose respectively; the 3 monosaccharide chromatographic peaks in the monosaccharide ion chromatogram of peach kernels represent fucose, galactose and glucose respectively.