CN114689750B - Construction method of HPLC fingerprint of n-butanol part of Mongolian almond and kindred species thereof - Google Patents
Construction method of HPLC fingerprint of n-butanol part of Mongolian almond and kindred species thereof Download PDFInfo
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Abstract
The invention relates to a construction method of HPLC fingerprint of a Mongolian almond and a kindred species n-butanol part thereof. The chromatographic conditions of the HPLC include that the chromatographic column is a C18 chromatographic column; mobile phase B is acetonitrile, mobile phase C is water; linear gradient elution was used to detect wavelength: 210nm to 225nm; the flow rate is 0.5-1.5 mL/min; column temperature: 30-35 ℃; the sample injection amount is 5-15 mu L. According to the invention, the HPLC fingerprint of the extract of the n-butanol part of the Mongolian almond and the kindred species thereof with good precision, stability and reproducibility is established for the first time, and the quality of the Mongolian almond and the kindred species thereof can be more comprehensively controlled by combining the component content measurement result.
Description
Technical Field
The invention belongs to the technical field of medicine detection, and particularly relates to a construction method of HPLC fingerprint of a Mongolian almond and a near-edge n-butanol part thereof.
Background
The almond (Amygdalus communis L.) is also called sweet almond, which is a deciduous tree or shrub of the amygdala of the Rosaceae, is originally produced in mountain areas of the middle and inferior regions, is distributed in the Xinjiang Uygur autonomous region, the inner Mongolian autonomous region, the Gansu province, the Shaanxi province and the like in China nowadays, is one of the famous woody oil tree species in the world, and belongs to the endangered protection plants in China. There are 6 species of wild species of Amygdalus communis (Amygdalus commacis l.), amygdalus mongolica (Amygdalus mongolica (maxim.) Ricker), amygdalus pedunculata (Amygdalus pedunculata pall. Amygdalus pedunculata), amygdalus elm (Amygdalus triloba (lindl.) Ricker), west Kang Biantao (Amygdalus tangutica (batal.) korsh. Tangut tonsil) and Amygdalus nana l. Condiner. The kernel can be used as a medicine instead of the bunge cherry kernel, has the effects of relieving cough, activating blood circulation, treating bronchitis, preventing cancer, relaxing bowel and promoting urination, and has remarkable effects on the aspects of antioxidation, blood fat reduction and the like. The health-care tea contains rich fatty oil (more than 50 percent), vitamins, amygdalin, protein, amino acid and the like, has extremely high nutritive value, can be used as medicine or health-care food, and is an excellent tree species integrating social and economic benefits.
The pharmacological actions of the Mongolian tonsils and the longpedunculate tonsils are greatly studied in the prior art: wu Tong and other researches show that the n-butanol extract of the amygdalus mongolica medicinal material has the effects of improving rat liver fibrosis and protecting liver; the research of the right Bovin and the like shows that the extract of the n-butyl alcohol part of the Mongolian almond medicinal material is an effective active part for resisting rat pulmonary fibrosis; research such as everolism and Liu Qing shows that the n-butanol extract of the amygdalus mongolica medicinal material can obviously improve rat kidney fibrosis. Zhao Yunshan and Bai Ying Chun are shown that the extracts of the n-butanol parts of the kernels of the medicinal materials of Mongolian amygdalus and Long pedunculata have the function of reducing blood fat, and amygdalin monomer compounds are extracted from the extracts for the first time.
The Chinese medicine has complex components, and it is important to comprehensively analyze and evaluate the consistency and stability of the medicine quality by using a certain method. The fingerprint spectrum of the traditional Chinese medicine is a comprehensive and quantifiable identification means, comprises identification methods such as High Performance Liquid Chromatography (HPLC), infrared spectrometry (IR), nuclear Magnetic Resonance (NMR) and the like, is widely applied to traditional Chinese medicine quality control, authenticity identification and spectral efficiency relation research, and plays a unique advantage in the fields. Therefore, the establishment of the fingerprint spectrum of the traditional Chinese medicine is significant for improving the quality of the traditional Chinese medicine and promoting the modern development of the traditional Chinese medicine. The HPLC fingerprint of the almond is established for the first time by the right Bovin and the like, which provides a certain method basis for evaluating the quality of the almond medicinal material, but researches the fingerprint of the active part which plays the medicinal effect.
Disclosure of Invention
The invention provides a more comprehensive experimental basis for the identification and quality control of almond medicinal materials, and provides a construction method of HPLC fingerprint of the n-butanol part of Mongolian almond and related species thereof.
According to the invention, 10 batches of almond medicinal materials of 7 production places of the inner Mongolia Baotou solid yang negligence chicken furrow, the inner Mongolia Baotou climbing elm, the inner Mongolia Baotou JiuFengshan, the inner Mongolia Baotou Zhao Changcheng, the Hebei province Zhuankou and the inner Mongolia Baotou city are taken as research objects, HPLC fingerprint research is further carried out on the kernel n-butyl alcohol part of the almond n-butyl alcohol part, and the similarity of the almond n-butyl alcohol part is analyzed, and meanwhile, the content of amygdalin in the almond n-butyl alcohol extract is measured by the method, so that the quality and the quality of Mongolia Baotou and near-edge medicinal materials of the almond are more scientifically, comprehensively and deeply evaluated.
The construction method of HPLC fingerprint of the n-butanol part of the Mongolian almond and the kindred species thereof is characterized by comprising the following steps:
1) Preparation of test solutions
Weighing almond kernel, grinding and sieving coarse powder, adding 5-15 times of ethanol, reflux-extracting, recovering ethanol from the extractive solution, and concentrating into soft extract; dissolving the soft extract in water, extracting the solution with petroleum ether, dichloromethane, ethyl acetate and n-butanol for 1-5 times, and concentrating the extractive solution to obtain extract of each part; weighing n-butanol extract, dissolving in methanol, ultrasonic treating, cooling, shaking, collecting solution, centrifuging, collecting supernatant, and filtering with microporous membrane to obtain sample solution of n-butanol extract of tonsil;
2) Preparation of standard solutions
Weighing amygdalin reference substance, dissolving with methanol, diluting, and shaking to obtain amygdalin reference substance solution; weighing a wild black cherry glycoside reference substance, dissolving with methanol, diluting, and shaking to obtain a wild black cherry glycoside standard substance solution;
3) HPLC chromatographic conditions
The chromatographic column is a C18 chromatographic column; the mobile phase is acetonitrile (B) -water (C); linear gradient elution was used to detect wavelength: 210nm to 225nm; the flow rate is 0.5-1.5 mL/min; column temperature: 30-35 ℃; the sample injection amount is 5-15 mu L.
Further, the specific method of the step 1) is as follows:
removing shell of almond kernel, grinding, and sieving with 24 mesh sieve; weighing 60g of coarse powder, adding 10 times of 95% ethanol, reflux-extracting for 2 times at 90 ℃,10 times of 70% ethanol, reflux-extracting for 1 time at 90 ℃ for 2 hours each time, combining 3 times of extracting solutions, recovering ethanol by a rotary evaporator, concentrating into thick extract, dissolving the thick extract and water in a volume of 1:1, extracting the solution for 3 times by using 2 times of petroleum ether, dichloromethane, ethyl acetate and n-butanol respectively, and concentrating the extracting solutions to dryness to obtain extracts of all parts; precisely weighing 25mg of n-butanol fraction extract, dissolving in methanol, standing to 5mL, performing ultrasonic treatment for 30min, cooling, shaking, collecting appropriate amount of solution, centrifuging, collecting supernatant, and filtering with 0.22 μm microporous membrane to obtain sample solution (5.0 mg/mL) of n-butanol fraction extract of tonsillitis.
Further, the specific method of the step 2) is as follows:
precisely weighing 25.0mg of amygdalin reference substance, placing in a 5mL volumetric flask, dissolving with methanol, diluting, fixing volume to 5mL, and shaking to obtain 5.0mg/mL amygdalin reference substance solution; accurately weighing 5.0mg of the wild black cherry glycoside reference substance, placing in a 2mL volumetric flask, dissolving with methanol, diluting, fixing volume to 2mL, and shaking to obtain 2.5mg/mL of wild black cherry glycoside reference substance solution.
Further, the specific method of the step 3) is as follows:
chromatographic column: agilent Eclipse XDB-C18 (4.6 nm. Times.250 nm,5 μm); mobile phase: acetonitrile (B) -water (C), eluting with a linear gradient; detection wavelength: 0 to 25min,210nm; 25-60 min,225nm; flow rate: 1.0mL/min; column temperature: 32 ℃; the sample injection amount was 10. Mu.L.
Further, the gradient elution procedure was:
further, the HPLC fingerprint of the n-butanol part of the Mongolian almond and the kindred species thereof totally detects 6 common peaks which are respectively No. 4 peak, no. 7 peak, no. 8 peak, no. 9 peak, no. 10 peak and No. 15 peak.
Further, the fingerprint of the n-butanol part of the amygdalin and the related species thereof has 6 characteristic common peaks, the chromatographic peak of the amygdalin is taken as a reference peak, the retention time is 1, and the relative retention time of other characteristic common peaks and the reference peak is respectively as follows: peak 4: 0.508+ -0.002; peak 7:0.894±0.001; peak 8:0.989±0.001; peak 9:1.00; peak 10: 1.164.+ -. 0.001; peak 15:2.116 + -0.004; wherein the No. 9 peak is amygdalin, and the No. 10 peak is pheomenine.
Further, the fingerprint of the n-butanol part of the amygdalin and the related species thereof has 6 characteristic common peaks, the chromatographic peak of the amygdalin is taken as a reference peak, the peak area is taken as 1, and the relative peak areas of other characteristic common peaks and the reference peak are respectively as follows: peak 4: 0.012+ -0.009; peak 7:0.059 + -0.011; peak 8:0.040±0.011; peak 9:1.00; peak 10: 0.123+ -0.080; peak 15: 0.074+ -0.060; wherein the No. 9 peak is amygdalin, and the No. 10 peak is pheomenine.
Advantageous effects
In recent years, the research on the fingerprint of the traditional Chinese medicine is very popular, but the research on the HPLC fingerprint of the n-butanol extract of the almond medicinal material is not reported. The method for determining and analyzing HPLC fingerprint of the Mongolian almond medicinal material and the n-butanol extract of the kindred species, which is established for the first time, has high precision, good stability, good repeatability and good separation degree of each chromatographic peak. The similarity evaluation result shows that the HPLC fingerprint similarity of the almond medicinal materials in different batches is higher (0.982-0.997), and the medicinal material quality is more stable. Meanwhile, by comparing 6 common peaks, 2 common peaks, namely amygdalin (peak 9) and pheasant (peak 10) were identified, wherein the unknown components of peak 4, peak 7, peak 8, peak 15 still need further identification analysis. Subsequently, the content of amygdalin in the extract was measured, which showed that the amygdalin content was 0.25-2.5 mg/mL (R 2 = 0.9991) exhibits good linearity; the average sample recovery rate is 99.4%; the RSD value was 1.03%.
The fingerprint has the characteristics of comprehensiveness and integrity, and the quality of the traditional Chinese medicine is comprehensively and deeply researched and controlled by utilizing the fingerprint, so that the stability of the quality of the traditional Chinese medicine in different batches is ensured, and the fingerprint has important significance for promoting the clinical application of the traditional Chinese medicine and promoting the modern development of the traditional Chinese medicine. According to the method, the HPLC fingerprint spectrum measuring method of the amygdalus mongolica medicinal material and the near-edge n-butanol extract thereof is established, the content of amygdalin in different batches of amygdalus mongolica kernel n-butanol extracts is measured by the method, the characteristics of effective medicinal components of the amygdalus mongolica medicinal material are further reflected, so that more comprehensive experimental basis is provided for the identification and the quality control of the amygdalus mongolica medicinal material, a foundation is laid for further scientific evaluation of the quality of the amygdalus mongolica medicinal material, and meanwhile, the improvement of an amygdalus mongolica medicinal material quality evaluation system is positively promoted.
According to the invention, the HPLC fingerprint of the extract of the n-butanol part of the Mongolian almond and the kindred species thereof with good precision, stability and reproducibility is established for the first time, and the quality of the Mongolian almond and the kindred species thereof can be more comprehensively controlled by combining the component content measurement result.
Drawings
FIG. 1 is an HPLC fingerprint of n-butanol portion of 10 batches of almond medicinal material.
FIG. 2 is a HPLC control fingerprint of n-butanol fraction of 10 batches of almond medicinal material.
Fig. 3 is a HPLC chromatogram of the mixed control.
Detailed Description
The present invention will be described in further detail with reference to the following specific embodiments, but the scope of the present invention is not limited to the specific examples.
Instrument for measuring and controlling the intensity of light
UltiMate 3000 high performance liquid chromatograph (PDA detector), cemerer Feier technology Co., ltd; agilent Eclipse XDB-C18 (4.6 nm. Times.250 nm,5 μm) column, agilent technologies Co., ltd; AP-01P vacuum pump, tianjin Ottsiamens instruments Co., ltd; KQ-500E ultrasonic cleaner, kunshan ultrasonic instruments Inc.; DZW-8-8 water bath, yongguangming medical instrument factory in Beijing city; RE-52A rotary evaporator, shanghai Asia Biochemical instruments factory; BSY-200T grinder, platinum European hardware Co., yongkang; an electronic analytical balance, shanghai Hengping instruments.
Medicine and reagent
10 batches of almond samples (S1-S10) are respectively collected from almond medicinal materials in different producing areas, and identified as dry mature seeds of the almond of the peach genus plant in the Rosaceae by the professor of Pinus lycopodii in the Baotou medical college of inner Mongolia science and technology university, and the source information is shown in table 1; amygdalin control (lot number: Z28A6L2815, purity greater than or equal to 98%, CAS number: 29883-15-6), wild black cherry glycoside control (lot number: X07J11L117770, purity greater than or equal to 98%, CAS number: 99-18-3), all purchased from Shanghai source leaf Biotechnology Co., ltd; acetonitrile (chromatographic purity, lot number 016880), methanol (chromatographic purity, lot number 016953) were all purchased from U.S. Mreda Technology Inc; petroleum ether, methylene dichloride, ethyl acetate and n-butanol are all analytically pure and purchased from Tianjin Kaitong chemical reagent Co.
TABLE 1 sources of tonsillitis drug samples
Example 1
1.1 preparation of sample solutions
Different batches of almond kernels were shelled, ground and sieved through a 24 mesh sieve. Weighing 60g of coarse powder, adding 10 times of 95% ethanol, reflux-extracting for 2 times at 90 ℃,10 times of 70% ethanol, reflux-extracting for 1 time at 90 ℃ for 2 hours each time, combining 3 times of extracting solutions, recovering ethanol by a rotary evaporator, concentrating into thick extract, dissolving the thick extract and water in a volume of 1:1, extracting the solution for 3 times by using 2 times of petroleum ether, dichloromethane, ethyl acetate and n-butanol each time, and concentrating each extracting solution to dryness to obtain 10 batches of almond extract. Precisely weighing 25mg of n-butanol part extract of each batch, adding methanol to dissolve and fix volume to 5mL, performing ultrasonic treatment for 30min, cooling, shaking uniformly, taking a proper amount of solution, centrifuging, taking supernatant, and filtering with a 0.22 μm microporous filter membrane to obtain 7 production places and 10 batches of sample solutions (5.0 mg/mL) of the n-butanol part extract of the almond medicinal material.
1.2 preparation of standard solutions
Precisely weighing 25.0mg of amygdalin reference substance, placing in a 5mL volumetric flask, dissolving with methanol, diluting, fixing volume to 5mL, and shaking to obtain 5.0mg/mL amygdalin reference substance solution; accurately weighing 5.0mg of the wild black cherry glycoside reference substance, placing in a 2mL volumetric flask, dissolving with methanol, diluting, fixing volume to 2mL, and shaking to obtain 2.5mg/mL of wild black cherry glycoside reference substance solution.
1.3HPLC chromatography conditions
Chromatographic column: agilent Eclipse XDB-C18 (4.6 nm. Times.250 nm,5 μm). Mobile phase: acetonitrile (B) -water (C) was eluted with a linear gradient and the procedure is shown in table 2. Detection wavelength: 0 to 25min,210nm; 25-60 min,225nm; flow rate: 1.0mL/min; column temperature: 32 ℃; the sample injection amount was 10. Mu.L.
TABLE 2 gradient elution procedure for mobile phases
1.4 data processing
And analyzing and establishing HPLC fingerprint of the almond n-butanol part extract by using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012.130723 version) and evaluating the similarity.
1.5 finger print methodology investigation
1.5.1 precision test sample solution (number: S4) of the same production place is taken, the sample is continuously injected for 6 times according to the chromatographic condition under the item of '1.3', the relative retention time RSD of each chromatographic peak is measured to be 0.01-0.21%, the relative peak area RSD is measured to be 0.29-2.10%, the similarity of each chromatographic peak map and the 'control fingerprint map' is measured to be 0.986-1.00, and the result shows that the instrument precision is good and meets the requirements.
1.5.2 stability test sample solutions (number: S4) newly prepared in the same place are taken, and are respectively sampled in 0, 2, 4, 8, 12 and 24 hours after preparation according to chromatographic conditions under the item of 1.3, so that the relative retention time RSD of each chromatographic peak in the same sample solution in 24 hours is measured to be 0.01-0.24%, the relative peak area RSD is 0.12-2.26%, the similarity of each chromatographic peak map and a contrast fingerprint map is 0.942-0.963%, and the result shows that the sample solution has good stability in 24 hours and meets the requirements.
1.5.3 repeatability test 6 parts of sample solution (number: S4) with the same batch number are taken, sample injection is carried out according to chromatographic conditions under the item of 1.3, detection analysis is carried out, the relative retention time RSD of each chromatographic peak in the 6 parts of samples is measured to be less than 0.02%, the RSD of the relative peak area is between 0.44 and 3.15%, the similarity RSD of each chromatographic peak pattern and the contrast fingerprint pattern is between 0.856 and 0.997%, and the result shows that the repeatability of the method is better.
1.6 establishment and analysis of fingerprint of n-butanol extract of amygdalus mongolica and related species thereof
Taking 10 batches of sample solutions of the n-butanol extract of the almond kernel, and respectively detecting according to the chromatographic conditions under the item of 1.3 to obtain HPLC chromatograms (S1-S10) of the n-butanol extract of the almond kernel of each batch, which is shown in figure 1. And converting the sample chromatograms of each batch into CDF format files, introducing software of a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012.130723 version) to calculate the similarity of each sample. The control fingerprint (R) was generated by automatic matching with a median method and a time window of 0.3, see fig. 2. The chromatographic peaks of 10 samples of almond n-butanol extract were observed and 6 common peaks were identified, of which amygdalin (peak 9) and pheomelanin (peak 10) were confirmed, see fig. 3. Amygdalin is one of main medicinal components of amygdalin, and the inventor adopts chromatography and spectrum means to conduct chemical component research on n-butanol part extract of amygdalin medicinal material in the early stage, and firstly identifies amygdalin monomer compound with the specific gravity of 47.72 percent. Since the occupied peak area was the largest among the common peaks, amygdalin (peak 9) was selected as a reference peak, and the retention time and the peak area were set to 1, and the relative retention time and the relative peak area of each common peak were calculated, respectively, and the results are shown in tables 3 and 4.
TABLE 3 relative retention times (min) of peaks common to HPLC finger prints of n-butanol extracts of different batches of almond samples
TABLE 4 relative peak areas (mAU) of common peaks of HPLC finger prints of n-butanol extracts of different batches of almond samples
1.7 evaluation of finger print similarity
HPLC chromatograms of n-butanol extracts of 10 batches of almond kernels are introduced into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012.130723 version), automatic matching is adopted, a median method is adopted to generate fingerprint of each sample and a reference fingerprint, the fingerprint is numbered, and then the similarity is calculated, and the result is shown in Table 5. The results show that the similarity of the fingerprint of each sample is 0.982-0.997 compared with the fingerprint of the control sample, which shows that the overall similarity of 10 batches of samples is higher, and the quality stability of the 10 batches of almond medicinal material kernel n-butanol extract medicinal material used in the experiment is better. Meanwhile, according to the fingerprint result (figure 1), 6 common peaks, namely a No. 4 peak, a No. 7 peak, a No. 8 peak, a No. 9 peak, a No. 10 peak and a No. 15 peak, are detected, wherein the No. 9 peak is amygdalin, and the No. 10 peak is pheomelanin. However, the chromatographic peaks of samples at different producing areas are slightly different, which indicates that the quality of the almond medicinal materials at different producing areas is different to a certain extent.
TABLE 5 similarity of n-butanol extracts for different batches of almond kernel samples
1.8 content determination methodology investigation
1.8.1 linear relation investigation and precise absorption of amygdalin standard substance solutions 2.5, 2.0, 1.0, 0.5 and 0.25mL under the item "1.2", respectively placing into 5mL volumetric flasks, diluting to scale marks with methanol solution, and shaking uniformly to obtain standard substance series solutions with dilution factors of 2, 2.5, 5, 10 and 20 times respectively. Sample injection and detection analysis are carried out according to the chromatographic condition under the item "1.3", a chromatogram is recorded, the peak area (y) is taken as an ordinate, the concentration (x, mg/mL) is taken as an abscissa, and linear regression analysis is carried out, and the result shows that the linear regression equation of amygdalin is y= 156.260x-6.821 (R) 2 = 0.9991), the concentrations exhibit good linear relationship in the range of 0.25 to 2.5 mg/mL.
1.8.2 precision test A proper amount (5.0 mg/mL) of amygdalin standard solution under item "1.2" was taken and the sample was continuously injected 6 times according to the chromatographic conditions under item "1.3". The RSD value of the amygdalin peak area is 0.39%, and the result shows that the method has good precision and meets the requirements.
1.8.3 repeatability test 6 parts of the sample solution (number: S4) with the same batch number are taken, sample injection is carried out according to the chromatographic condition under the item "1.3", and detection analysis is carried out, so that the RSD value of the amygdalin peak area is 1.29%, and the result shows that the content determination method is good in repeatability.
1.8.4 the stability test is to take a newly prepared sample solution (number: S4) of the same production place, sample injection is carried out at 0, 2, 4, 8, 12 and 24 hours after preparation according to the chromatographic conditions under the item of 1.3, the RSD value of the peak area of amygdalin in 24 hours is 1.37%, and the result shows that the amygdalin has good stability in 24 hours and meets the requirements.
1.8.5 sample recovery test 1mL of a sample solution (number: S4) with known content is precisely measured, 9 parts are added, proper amounts of amygdalin standard substance solution are respectively added to prepare low, medium and high concentration mixed solutions, 3 parts of mixed solutions are prepared in parallel for each concentration, sample injection is performed according to chromatographic conditions under the item "1.3", peak areas are recorded, and sample recovery is calculated, and the result is shown in Table 6.
TABLE 6 sample recovery test results
1.8.6 sample content determination A proper amount of n-butanol extract sample of each batch of almond medicinal material is taken, a sample solution to be tested is prepared according to the item "1.2", and then sample injection, detection and analysis are carried out according to the chromatographic condition under the item "1.3", and the parallel determination is carried out for 3 times. The experimental results were statistically analyzed by SPSS 19.0, the data are expressed as + -s, and the results of measurement of the amygdalin sample content are shown in Table 7.
Table 7 content of amygdalin in extract of amygdalin and related species of amygdalin
Example 2:
comparison of the separation effect of two mobile phases, methanol-water and acetonitrile-water, the column was subjected to the following conditions: agilent Eclipse XDB-C18 (4.6 nm. Times.250 nm,5 μm); mobile phase: methanol (B) -water (C)/acetonitrile (B) -water (C), using linear gradient elution, detection wavelength: 210nm; flow rate: 1.0mL/min; column temperature: 30 ℃; the sample injection amount was 10. Mu.L.
The gradient elution procedure was:
as a result, when methanol-water is used as the mobile phase, the chromatographic peak has more peaks with steep front and gentle rear, and asymmetric tail peaks and components are not completely separated and overlapped. When acetonitrile-water is used as a mobile phase, the chromatographic peaks have more peaks, good symmetry, ideal peak height and peak area, and obvious separation effect among the peaks, so that the acetonitrile-water can be used as the mobile phase to better separate and identify the components in the n-butanol part extract of the almond medicinal material.
Example 3:
comparing chromatograms (210 nm, 225nm, 250 nm) of n-butanol extracts of seed kernels of the amygdalus medicinal materials at different wavelengths, and performing chromatographic column under the following conditions: agilent Eclipse XDB-C18 (4.6 nm. Times.250 nm,5 μm); mobile phase: acetonitrile (B) -water (C), eluting with linear gradient, and detecting wavelengths respectively as follows: 210nm/225nm/250 nm/0-25 min,210nm; 25-60 min,225nm; flow rate: 1.0mL/min; column temperature: 30 ℃; the sample injection amount was 10. Mu.L.
As a result, the chromatographic peak under the single wavelength condition has more tailing peaks and relatively short and fat steamed bread peaks, while the chromatographic peak has stable baseline, maximum absorption intensity and maximum peak output under the condition of wavelength switching between 210 and 225nm, and has better peak height, peak area and peak shape, obvious separation effect and no mutual interference. Therefore, the detection is carried out under the condition of wavelength switching between 210nm and 225nm, and the components in the n-butanol part extract of the almond medicinal material can be better separated and identified.
Example 4
The effect of different column temperatures (30 ℃, 32 ℃, 35 ℃) on the chromatographic peaks of different batches of test substances is compared, and the chromatographic column is subjected to the following conditions: agilent Eclipse XDB-C18 (4.6 nm. Times.250 nm,5 μm); mobile phase: acetonitrile (B) -water (C), eluting with linear gradient, and detecting wavelengths respectively as follows: 0 to 25min,210nm; 25-60 min,225nm; flow rate: 1.0mL/min; column temperature: 30 ℃/32 ℃/35 ℃; the sample injection amount was 10. Mu.L.
The results show that the chromatograms at the column temperatures have little overall difference and have more peaks, but the overall shapes, heights, areas, separation degrees and the like of chromatographic peaks are relatively good at the column temperature of 32 ℃. Therefore, the final detection temperature of 32 ℃ is selected, and the components in the n-butanol part extract of the almond medicinal material can be better separated and identified.
The above embodiments are only for illustrating the present invention, and are not limiting of the present invention. While the invention has been described in detail with reference to the embodiments, those skilled in the art will appreciate that various combinations, modifications, and substitutions can be made thereto without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (5)
1. The construction method of HPLC fingerprint of the n-butanol part of the Mongolian almond and the kindred species thereof is characterized by comprising the following steps:
1) Preparation of test solutions
Removing shell of almond kernel, grinding, and sieving with 24 mesh sieve; weighing coarse powder 60g, adding 10 times of 95% ethanol, reflux-extracting for 2 times at 90 ℃,10 times of 70% ethanol, reflux-extracting for 1 time at 90 ℃, 2h each time, combining 3 times of extracting solutions, recovering ethanol by a rotary evaporator, concentrating into thick extract, dissolving the thick extract and water in 1:1 volume, extracting the solution for 3 times by using 2 times of petroleum ether, dichloromethane, ethyl acetate and n-butanol respectively, and concentrating the extracting solutions to dryness to obtain extracts of all parts; precisely weighing n-butanol extract 25mg, dissolving in methanol and fixing volume to 5mL, performing ultrasonic treatment for 30min, cooling, shaking, collecting appropriate amount of solution, centrifuging, collecting supernatant, and filtering with 0.22 μm microporous membrane to obtain sample solution of n-butanol extract of tonsillitis medicine with concentration of 5.0 mg/mL;
2) Preparation of standard solutions
Weighing amygdalin reference substance, dissolving with methanol, diluting, and shaking to obtain amygdalin reference substance solution; weighing a wild black cherry glycoside reference substance, dissolving with methanol, diluting, and shaking to obtain a wild black cherry glycoside standard substance solution;
3) HPLC chromatographic conditions
The chromatographic column is a C18 chromatographic column; mobile phase B is acetonitrile, mobile phase C is water; linear gradient elution was used to detect wavelength: 210 nm-225 nm; the flow rate is 0.5-1.5 mL/min; column temperature: 30-35 ℃; the sample injection amount is 5-15 mu L;
the Mongolian almond and its kindred seeds are selected from Mongolian almondAmygdalus mongolica (Maxim.) RickerAlmond with long pedunclesAmygdalus pedunculata Pall.Or elm Amygdalus triloba (lindl.) Ricker;
the specific method of the step 3) is as follows:
chromatographic column: agilent Eclipse XDB-C18,4.6 mm ×250 mm,5 μm; the mobile phase B is acetonitrile, the mobile phase C is water, and linear gradient elution is adopted; detection wavelength: 0-25 min,210nm; 25-60 min,225nm; flow rate: 1.0mL/min; column temperature: 32 ℃; the sample injection amount is 10 mu L;
the gradient elution procedure was:
。
2. The construction method according to claim 1, wherein the specific method of step 2) is as follows:
precisely weighing amygdalin reference 25.0mg, placing in 5mL volumetric flask, dissolving with methanol, diluting, fixing volume to 5mL, and shaking to obtain amygdalin reference solution of 5.0 mg/mL; accurately weighing the reference sample 5.0. 5.0mg of the wild black cherry glycoside, placing the reference sample in a 2mL volumetric flask, dissolving the reference sample in methanol, diluting the reference sample to 2mL, and shaking the solution uniformly to obtain 2.5mg/mL of the reference sample solution of the wild black cherry glycoside.
3. The method according to any one of claims 1-2, wherein the HPLC fingerprint of the n-butanol portion of the Mongolian almond and the kindred species thereof detects 6 common peaks, which are respectively No. 4 peaks, no. 7 peaks, no. 8 peaks, no. 9 peaks, no. 10 peaks and No. 15 peaks.
4. The construction method according to claim 3, wherein the fingerprint of the n-butanol portion of the amygdalin and the related species thereof has 6 characteristic common peaks, the chromatographic peak of amygdalin is taken as a reference peak, and the retention time is taken as 1, and the relative retention time of the other characteristic common peaks and the reference peak is respectively as follows: peak 4: 0.508+ -0.002; peak 7:0.894±0.001; peak 8:0.989±0.001; peak 9:1.00; peak 10: 1.164.+ -. 0.001; peak 15:2.116 + -0.004; wherein the No. 9 peak is amygdalin, and the No. 10 peak is pheomenine.
5. The construction method according to claim 3, wherein the fingerprint of the n-butanol portion of the amygdalin and the related species thereof has 6 characteristic common peaks, the chromatographic peak of amygdalin is taken as a reference peak, and the peak area is taken as 1, and the relative peak areas of the other characteristic common peaks and the reference peak are respectively as follows: peak 4: 0.012+ -0.009; peak 7:0.059 + -0.011; peak 8:0.040±0.011; peak 9:1.00; peak 10: 0.123+ -0.080; peak 15: 0.074+ -0.060; wherein the No. 9 peak is amygdalin, and the No. 10 peak is pheomenine.
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| Chromatographic Determination of Cyanoglycosides Prunasin and Amygdalin in Plant Extracts Using a Porous Graphitic Carbon Column;V. BERENGUER-NAVARRO 等;J. Agric. Food Chem.;第50卷;6960-6963 * |
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