Construction method and application of trichosanthes kirilowii maxim polysaccharide fingerprint spectrum

Abstract

The invention relates to the technical field of traditional Chinese medicine quality analysis and detection, in particular to a construction method and application of a trichosanthes kirilowii maxim polysaccharide fingerprint spectrum. According to the method, trichosanthes kirilowii maxim polysaccharide is obtained in an ultrasonic extraction and alcohol precipitation mode, then the trichosanthes kirilowii maxim polysaccharide is depolymerized through an ultrasonic-assisted acidolysis method, trichosanthes kirilowii maxim polysaccharide depolymerized matter is subjected to hydrophilic interaction chromatography-electrospray detector-electrospray-quadrupole-time-of-flight mass spectrometry (HILIC-CAD / ESI-Q-TOF / MS) analysis, and 16 chemical components are obtained in total. 16 chemical components are used as common peaks to establish the trichosanthes kirilowii maxim polysaccharide fingerprint spectrum, and a method and technical support are provided for trichosanthes kirilowii maxim polysaccharide quality evaluation and control.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of quality analysis and detection of traditional Chinese medicine, and particularly relates to a construction method and application of a Gualou polysaccharide fingerprint. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing an understanding of the background of the present application and should not be taken as an acknowledgement or any form of suggestion that this information forms prior art with respect to any patentable matter disclosed in this application.

[0003] Gualou is the dried mature fruit of Trichosanthes kirilowii Maxim or Trichosanthes rosthornii Harms; Gualou is one of traditional Chinese medicines; modern medical research shows that Gualou extract has multiple effects such as expanding coronary artery, increasing blood flow, improving hypoxia tolerance, reducing serum cholesterol, antibacterial, anticancer and the like.

[0004] Different parts of Gualou can be used as medicine, which are Gualou (fruit), Gualoupi (pericarp), Gualouzi (seed) and Tianhuafen (root), and the chemical components contained in each part are also not completely the same, mainly including oil, organic acids, proteins, amino acids and sugar components.

[0005] Polysaccharide is polymerized by 10 or more monosaccharide molecules through glycosidic bond, has a large molecular weight, generally tens of thousands or even millions, and is one of four basic substances constituting life. Polysaccharide is one of the most representative bioactive components in Gualou, and has multiple pharmacological activities such as antioxidant, anticoagulant, hypoglycemic, antitumor, immunomodulatory, liver protection and the like. However, due to the complexity of polysaccharide structure, there are few reports on the analysis and quality control of Gualou polysaccharide.

[0006] At present, the phenol-sulfuric acid method or the anthrone-sulfuric acid method is mainly used in the determination of polysaccharide content in the first volume of the 2020 edition of Chinese Pharmacopoeia for the quality control of traditional Chinese medicine polysaccharide, and lacks specificity. In addition, postharvest drying is an important link in the formation of the quality of traditional Chinese medicine, and the drying method has a significant influence on the monosaccharide composition, molecular weight, conformation and biological activity of polysaccharide. Due to the lack of specific and effective quality control method of Gualou polysaccharide, the postharvest drying process is blind and arbitrary, which seriously affects the quality of Gualou polysaccharide. SUMMARY

[0007] In order to overcome the above problems, the present application provides a construction method and application of a Gualou polysaccharide fingerprint.

[0008] In order to achieve the above technical purposes, the present application adopts the following technical solutions: In a first aspect, the present application provides a method for constructing a fingerprint of Trichosanthes kirilowii polysaccharide, comprising the following steps: (1) crushing and sieving the dried Trichosanthes kirilowii to obtain Trichosanthes kirilowii powder; (2) adding deionized water to the Trichosanthes kirilowii powder, ultrasonic extraction, filtering to collect the filtrate, alcohol precipitation, and centrifugal collection of the precipitate, which is the Trichosanthes kirilowii polysaccharide; (3) adding trifluoroacetic acid to the Trichosanthes kirilowii polysaccharide after re-dissolving, ultrasonic-assisted acid hydrolysis, nitrogen blowing dry, re-dissolving in acetonitrile-water mixed solution to obtain a test solution; (4) injecting the test solution into a hydrophilic interaction chromatography-electrospray detector-electrospray-quadrupole-time-of-flight mass spectrometer (HILIC-CAD / ESI-Q-TOF / MS) for detection to obtain 16 chemical components; (5) taking the 16 chemical components as common peaks to establish a fingerprint of Trichosanthes kirilowii polysaccharide.

[0009] In one or more embodiments, the drying method comprises shade drying, oven drying, freeze drying, and microwave drying, preferably microwave drying.

[0010] Preferably, the temperature for oven drying is 50-90 ℃. Preferably, the freeze drying method comprises pre-freezing at -18-25 ℃ for 10-14 h in a refrigerator, and then placing in a vacuum freeze dryer for freeze drying under the conditions of cold trap -35--45 ℃ and vacuum degree 0.8-1.2 Pa until constant weight.

[0011] Preferably, the microwave drying conditions are 300-700 W for 15-30 min.

[0012] In one or more embodiments, the Trichosanthes kirilowii comprises whole Trichosanthes kirilowii, Trichosanthes kirilowii peel, Trichosanthes kirilowii pulp, and Trichosanthes kirilowii seeds, preferably whole Trichosanthes kirilowii or Trichosanthes kirilowii peel, and further preferably Trichosanthes kirilowii peel.

[0013] In one or more embodiments, the dried Trichosanthes kirilowii is crushed and sieved through a 50-80 mesh sieve, preferably a 60 mesh sieve.

[0014] In one or more embodiments, in step (2), the mass ratio of Trichosanthes kirilowii powder to deionized water is 1:(8-12), preferably 1:10.

[0015] In one or more embodiments, in step (2), the ultrasonic extraction temperature is 85-100 ℃, preferably 90 ℃; the ultrasonic extraction power is 300-500 w, preferably 350 w; and the ultrasonic extraction time is 20-30 min, preferably 25 min.

[0016] In one or more embodiments, in step (2), the temperature of the alcohol precipitation is 0-6 °C, preferably 4 °C; the time of the alcohol precipitation is 10-14 h, preferably 12 h.

[0017] In one or more embodiments, in step (3), the re-dissolving conditions include: the solvent is deionized water; the temperature of the re-dissolving is 80-100 °C.

[0018] In one or more embodiments, in step (3), the temperature of the acidolysis is 70-100 °C, preferably 80 °C; the time of the acidolysis is 10-30 min, preferably 15 min; the power of the ultrasonic during the acidolysis is 240-360 w, preferably 280 w; the concentration of the trifluoroacetic acid during the acidolysis is 3-6 mol / L, preferably 4 mol / L.

[0019] In one or more embodiments, in step (3), the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is (2.5-4):1, preferably 3:1.

[0020] In one or more embodiments, in step (4), the chromatographic conditions are: The chromatographic column is Waters Xbridge Amide column; the mobile phase A is acetonitrile, and the mobile phase B is 0.8% formic acid water, gradient elution.

[0021] Preferably, the gradient elution conditions include: 0-12 min, 92%-85% A; 12-30 min, 85%-84% A; 30-40 min, 84%-75% A; 40-60 min, 75%-50% A.

[0022] Preferably, during the detection, the flow rate is 0.7-1 mL / min, preferably 0.8 mL / min.

[0023] Preferably, during the detection, the injection volume is 18-22 μL, preferably 20 μL; Preferably, during the detection, the column temperature of the chromatographic column is 35-45 °C, preferably 40 °C.

[0024] In one or more embodiments, in step (4), the detection parameters of the electrospray detector include: The gas source is N2; the pressure is 8.8 kPa; Filter 2.0 sec, atomizer temperature 60 °C.

[0025] In one or more embodiments, in step (4), the mass spectrometry conditions are: The instrument was operated in positive and negative ionization modes respectively; the operation parameters were as follows: the pressure of the atomizer was 200 kPa, the flow rate of the dry gas was 10.0 L·min -1 , the temperature of the dry gas was 350 ℃, the cracking voltage was 10 eV, the capillary voltage in positive and negative ion modes was 3.5 kV and 3.0 kV respectively, and the scanning range was m / z 50~2000.

[0026] In one or more embodiments, in step (4), the 16 chemical components are peak1, peak2, peak3, peak4, peak5, peak6, peak7, peak8, peak9, peak10, peak11 and peak12 respectively. The retention time of peak1 is 7.3~7.7 min; the retention time of peak2 is 8.8~9.2 min; the retention time of peak3 is 9.6~10.0 min; the retention time of peak4 is 11.0~11.4 min; the retention time of peak5 is 13.8~14.2 min; the retention time of peak6 is 17.5~17.7 min; the retention time of peak7 is 19.1~19.5 min; the retention time of peak8 is 19.8~20.2 min; the retention time of peak9 is 21.1~21.5 min; the retention time of peak10 is 26.9~27.3 min; the retention time of peak11 is 40.4~40.8 min; the retention time of peak12 is 48.8~49.2 min; the retention time of peak13 is 51.0~51.4 min; the retention time of peak14 is 52.4~52.8 min; the retention time of peak15 is 55.1~55.5 min; and the retention time of peak16 is 58.0~58.4 min.

[0027] In a fourth aspect of the present application, the application provides the use of the gualou polysaccharide fingerprint constructed by the construction method of the first aspect in quality evaluation and control.

[0028] The present application has the following advantages: (1) The present application uses ultrasonic extraction and alcohol precipitation to obtain gualou polysaccharide, and then uses ultrasonic-assisted acidolysis to depolymerize the gualou polysaccharide. The gualou polysaccharide depolymerate is analyzed by hydrophilic interaction chromatography-electrospray detector-electrospray-quadrupole-time-of-flight mass spectrometry (HILIC-CAD / ESI-Q-TOF / MS), and 16 chemical components are obtained. The 16 chemical components are used as common peaks to establish a gualou polysaccharide fingerprint, which provides a method and technical support for the quality evaluation and control of gualou polysaccharide.

[0029] (2) The present application realizes the correct differentiation of different medicinal parts (whole Trichosanthes fruit, Trichosanthes fruit peel, Trichosanthes fruit seed and Trichosanthes fruit core) of Trichosanthes fruit and the differentiation of Trichosanthes fruit polysaccharides treated by different drying methods through Trichosanthes fruit polysaccharide fingerprint combined with multivariate statistical analysis, and peak 11 (unknown), peak 10 (unknown), peak 1 (unknown), peak 8 (glucose), peak 15 (DP2) and peak 5 (galactose) can be used as differentiation markers. BRIEF DESCRIPTION OF DRAWINGS

[0030] The accompanying drawings, which form a part of the present description, are used to provide further understanding of the present application, and the illustrative embodiments of the present application and their description serve the purpose of explanations rather than limiting the present application.

[0031] Figure 1 is a HILIC-CAD chromatogram of Trichosanthes fruit polysaccharide; Figure 2 is a peak area change graph of representative peaks of Trichosanthes fruit polysaccharide under different conditions, wherein A is acid hydrolysis concentration, B is acid hydrolysis power, C is acid hydrolysis temperature and D is acid hydrolysis time; Figure 3 is a peak 7 MS graph under negative ion mode; Figure 4 is a DP2 MS graph (A) and MS / MS graph (B) under negative ion mode; Figure 5 is a HILIC-CAD chromatogram of Trichosanthes fruit polysaccharide of different parts (whole Trichosanthes fruit, Trichosanthes fruit peel, Trichosanthes fruit seed and Trichosanthes fruit core); Figure 6 is a principal component analysis score graph of Trichosanthes fruit polysaccharide fingerprint of different parts; Figure 7 is a depolymerization product fingerprint graph of Trichosanthes fruit polysaccharide treated by different drying methods; Figure 8 is a peak area change graph of representative polysaccharide hydrolysis peaks of Trichosanthes fruit under different drying methods; Figure 9 is a multivariate statistical analysis graph of Trichosanthes fruit polysaccharide fingerprint under different drying methods, wherein A is a PCA score graph, B is a PLS-DA score graph, C is a 200 times permutation test cross validation graph and D is a VIP graph (VIP>1.0). DETAILED DESCRIPTION

[0032] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0033] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, components, and / or groups thereof, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0034] In order for those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in conjunction with specific examples.

[0035] The gualou used in the following examples was picked from the Pingyin planting base of Jinan Hobo Traditional Chinese Medicine Material Co., Ltd. and identified by Qilu University of Technology (Shandong Academy of Sciences) as the mature fruit of Trichosanthes kirilowii Maxim of the Cucurbitaceae family.

[0036] Example 1 (1) Drying method of gualou: Shadow drying group (SD): placed in a cool indoor place and naturally dried to constant weight; Drying group (HD): select two groups and place them in 60 ℃ and 80 ℃ air oven for intermittent drying, take them out every 12 h, dry them at room temperature for 12 h, and then continue hot air drying until constant weight to prevent gualou from cracking caused by continuous high temperature, and mark them as HD-60 and HD-80 respectively; Freeze-drying group (FD): first pre-freeze for 12 h in a-20 ℃ refrigerator, then place it in a vacuum freeze dryer, and freeze-dry it to constant weight under the conditions of cold trap-40 ℃ and vacuum degree 1 Pa; Microwave drying group (MD): select two groups for microwave drying, set the power to 400 W and 600 W respectively for 20 min to avoid gualou deterioration caused by long baking time, and mark them as MD-400 and MD-600 respectively; After drying, the skin, seeds, and pulp of gualou treated by different methods are separated, and the pulp wrapped outside the seeds and the seeds are washed and dried. The whole gualou, gualou skin, gualou seeds, and gualou pulp treated by drying are crushed through a 60-mesh sieve for use.

[0037] (2) Extraction and ultrasonic-assisted acid hydrolysis of gualou polysaccharides: Extraction: 1.0 g of snake gourd sample powder was precisely weighed into a stoppered triangular flask, 10 mL of deionized water at 90 ℃ was added, and it was extracted in an ultrasonic extraction instrument (350 W) for 25 min. The supernatant was filtered, the filtrate was added with 95% (volume fraction) ethanol, and it was placed in a refrigerator at 4 ℃ for alcohol precipitation for 12 h, and then centrifuged (4500 g) for 15 min. The precipitate was dried by evaporation in a water bath at 80 ℃, and snake gourd polysaccharide was obtained. Then it was redissolved with 5 mL of hot water, transferred to a 10 mL volumetric flask, and diluted to volume with water.

[0038] Ultrasonic-assisted acid hydrolysis: 700 μL of the sample after dilution was added to trifluoroacetic acid for ultrasonic-assisted acid hydrolysis. After nitrogen blowing and drying, it was redissolved in 1 mL of acetonitrile-water mixed solution (volume ratio: acetonitrile: water = 3:1) to obtain a test sample solution. After filtration through a 0.22 μm filter membrane, it was stored in a refrigerator at 4 ℃ for standby use.

[0039] The conditions of ultrasonic-assisted acid hydrolysis were optimized, and the acid hydrolysis concentration (TFA: 3, 4, 5, 6 mol·L -1 ), acid hydrolysis power (245, 280, 315, 350 W), acid hydrolysis time (10, 15, 20, 25, 30 min), and acid hydrolysis temperature (70, 80, 90 ℃) were investigated.

[0040] (3) Chromatography-mass spectrometry analysis conditions Chromatography conditions: Unimates 3000 high-performance liquid chromatograph (ThermoFisher Scientific, USA). Specifically: chromatography column: Waters Xbridge Amide column (4.6 mm×250 mm, 5 μm, Waters, USA); mobile phase A: acetonitrile solution, B: 0.8% formic acid water; gradient elution, gradient elution conditions include: 0~12 min, 92%~85% A; 12~30 min, 85%~84% A; 30~40 min, 84%~75% A; 40~60 min, 75%~50% A. Flow rate 0.8 mL·min -1 ; injection volume 20 μL; column temperature 40 °C.

[0041] The Charged Aerosol Detection (CAD) detector can analyze compounds with no / weak ultraviolet absorption, has high sensitivity and response consistency, and is an effective supplement to ultraviolet detection. The CAD detection parameters are as follows: gas source is N2, pressure is 8.8 kPa, Filter is 2.0 sec, and atomizer temperature is 60 °C.

[0042] Mass spectrometry conditions: IMPACT II electrospray time-of-flight mass spectrometer (ESI-Q-TOF / MS) (Bruker, Germany) was used for MS and MS / MS data acquisition and analysis. The instrument was operated in positive and negative ionization modes, respectively, with the following operating parameters: spray pressure 200 kPa, drying gas flow rate 10.0 L·min -1 , drying gas temperature 350 ℃, fragmentation voltage 10 eV, capillary voltage positive ion and negative ion mode 3.5 kV, 3.0 kV, respectively, and scan range m / z 50-2000. The data collected by mass spectrometry were analyzed and processed using Data Analysis software (Bruker, Germany).

[0043] Example 2 (1) Chromatographic condition optimization: The polar hydrolysate of cucumis melo polysaccharide has certain difficulty in chromatographic analysis. In the present application, various chromatographic conditions are tested and adjusted, including the type of chromatographic column used, the selection of mobile phase system, volume flow rate, column temperature and extraction, to obtain the best chromatographic separation effect.

[0044] Waters Xbridge Amide column (4.6 mm x 250 mm, 5 μm) and Waters Symmetry C18 chromatographic column (4.6 mm x 250 mm, 5.0 μm) were compared. The results showed that Waters Xbridge Amide column (4.6 mm x 250 mm, 5 μm) had better separation effect on polysaccharide, more chromatographic peaks and better peak symmetry, and therefore was used for further analysis.

[0045] At the same time, it was found that acetonitrile aqueous solution was more suitable for HILIC chromatographic column than other polar solvents, with more chromatographic peaks and better separation, so acetonitrile and water were selected as the separation mobile phase. When only acetonitrile and water were used in the mobile phase, most of the target peaks showed tailing phenomenon. Therefore, different proportions of formic acid (0.2 %, 0.4 %, 0.6 %, 0.8 %, 1.0 %) were added to the water phase. The results showed that acetonitrile-0.8 % formic acid aqueous system had better peak symmetry and sharp peak shape. Similarly, the effects of different flow rates (0.6, 0.8, 1.0 mL·min -1 ) and different column temperatures (20, 30, 40 ℃) on chromatographic peaks were investigated. The experiment finally determined that acetonitrile-0.8 % formic acid aqueous solution, 0.8 mL·min-1 flow rate and 40 ℃ column temperature could obtain chromatogram with good separation degree, stable baseline and symmetrical peak shape. Figure 1

[0046] (2) Optimization of ultrasonic-assisted acid hydrolysis conditions:​ Because of the complex composition of the Trichosanthes sample, in order to further improve the depolymerization efficiency of polysaccharides, the acid hydrolysis concentration (TFA: 3, 4, 5, 6 mol·L -1 ), acid hydrolysis power (245, 280, 315, 350 W), acid hydrolysis time (10, 15, 20, 25, 30 min) and acid hydrolysis temperature (70, 80, 90 ℃) were optimized. Six chromatographic peaks were selected for single factor experiment analysis, and the results are shown in Figure 2 . As can be seen from Figure 2 , when the acid hydrolysis concentration is 4 mol·L -1 , the acid hydrolysis power is 280 W, the acid hydrolysis temperature is 80 ℃, and the acid hydrolysis time is 25 min, the polysaccharide peak area has been significantly improved. Therefore, the experimental conditions should be strictly controlled. Lower acid concentration and hydrolysis temperature may lead to low depolymerization efficiency, but higher acid concentration and hydrolysis temperature may lead to complete hydrolysis of specific polysaccharides and many side reactions.

[0047] (3) HILIC-CAD / ESI-Q-TOF / MS analysis of Trichosanthes polysaccharide depolymerization The HILIC eluent was connected to the ESI-Q-TOF-MS instrument, and the accurate molecular weight and secondary ion analysis were carried out under negative ion mode. Through HILIC-ESI-Q-TOF / MS analysis, a variety of monosaccharides and oligosaccharides were identified from Trichosanthes polysaccharide depolymerization. By comparing the chromatographic retention time and accurate mass information of monosaccharide standard, the monosaccharides in the hydrolyzate were accurately identified. By analyzing the accurate molecular weight information and secondary fragment ion information of the chromatographic peak, the oligosaccharides were preliminarily identified, and the results are shown in Table 3.

[0048] Table 3 Mass spectrum identification of Trichosanthes polysaccharide based on HPLC-ESI-Q-TOF / MS

[0049] peak7 The MS mass spectrum of the quasi-molecular ion peak [M-H]- 179.0548 produced M / Z 161.0451 [M-H-18]- fragment ion, by comparing the mass difference between the two fragment ions and the quasi-molecular ion and combining the multi-stage mass spectrum analysis, it can be determined that the 161.0451 fragment ion is formed by dehydration of the quasi-molecular ion, and by combining the chromatogram of the corresponding monosaccharide standard, it can be determined that this substance is fructose, and the mass spectrum is shown in Figure 3 .

[0050] In negative ion full scan mode, DP2 produced quasi-molecular ion peak M / Z 340.9456 [M-H] - and [M+COO-]- M / Z 384.9632, the possible molecular formula is C 12 H 22 O 11 Further analysis by secondary scanning, the glycosidic bond is broken, the parent ion loses the glycosyl C6H 12 O6and combines with the solution COO - , forming M / Z 226.9654 [M-C6H 12 O6+COO] - , thus deducing that this substance is DP2, the primary and secondary mass spectra are shown in Figure 4 .

[0051] (4) Comparison of different parts of Trichosanthes kirilowii polysaccharide fingerprints The polysaccharide depolymer of different medicinal parts of Trichosanthes kirilowii was analyzed, and the results are shown in Figure 5 . From Figure 5 , it can be observed that the peak area response value of each part of the whole Trichosanthes kirilowii, Trichosanthes kirilowii peel, Trichosanthes kirilowii seed, and Trichosanthes kirilowii pulp is the highest in Trichosanthes kirilowii peel, followed by the whole Trichosanthes kirilowii, and the Trichosanthes kirilowii seed has the lowest response. In addition, from Figure 5 , it can also be seen that the number of chromatographic peaks in Trichosanthes kirilowii seed is significantly less than that in the whole Trichosanthes kirilowii and Trichosanthes kirilowii peel. This may be due to the fact that Trichosanthes kirilowii seed is rich in higher fatty components and has less sugar content, and the monosaccharide composition may also be different, so many monosaccharide chromatographic peaks cannot be detected in the seed. From Figure 5 , it can also be seen that there are also a small amount of chromatographic peaks that cannot be detected in Trichosanthes kirilowii pulp. The above results show that the ultrasonic-assisted acid hydrolysis-HILIC-CAD method can reflect the structural differences of polysaccharides in different medicinal parts of Trichosanthes kirilowii to some extent.

[0052] The polysaccharide depolymer fingerprint analysis method was used to analyze the polysaccharides of different parts (whole Trichosanthes kirilowii, Trichosanthes kirilowii peel, Trichosanthes kirilowii seed, and Trichosanthes kirilowii pulp) of Trichosanthes kirilowii, and multivariate statistical analysis method was used to classify the polysaccharide samples of different parts of Trichosanthes kirilowii, and the results are shown in Figure 6 . The main characteristics of the polysaccharide depolymer fingerprint are represented by the cumulative contribution rate, and the cumulative contribution rate of the first two principal components (PC1, PC2) is (80.9%). From Figure 6 , it can be seen that the polysaccharide depolymer fingerprint combined with principal component analysis can correctly distinguish different medicinal parts. The score plots of different medicinal parts are located in four quadrants, and the score plots of the same part are close, indicating that this method can well distinguish different medicinal parts of Trichosanthes kirilowii. In addition, the whole Trichosanthes kirilowii and Trichosanthes kirilowii peel are close, while they are far away from Trichosanthes kirilowii seed and pulp, further revealing the differences in the structure and content of polysaccharides in different parts of Trichosanthes kirilowii.

[0053] (5) Effect of different drying methods on the polysaccharide fingerprint of Trichosanthes kirilowii The effects of different postharvest processing methods on Trichosanthes kirilowii polysaccharide were investigated using the fingerprint analysis method. Figure 7 ).from Figure 7 As can be seen, the fingerprint profiles of Trichosanthes kirilowii polysaccharide hydrolysates are consistent under different drying methods, but the intensities of each chromatographic peak differ to some extent. To further investigate the changes of Trichosanthes kirilowii polysaccharide under different drying methods, the peak areas of several representative chromatographic peaks were selected to study the effect of different drying methods on Trichosanthes kirilowii polysaccharide. Figure 8 Among the four selected peaks, peak 5 (galactose) had the highest peak area, followed by peak 15 (DP2) and peak 16 (DP5), while peak 8 (glucose) had the lowest. Of the four drying methods, air drying (SD) produced the highest DP5 content and the lowest peak 5 (galactose) content; however, considering its long drying time, this method is not the optimal choice. Using the oven drying (HD) method at 60 ℃ and 80 ℃, the contents of peak 8 (glucose) and peak 16 (DP5) were the lowest, indicating that this method has a potential destructive effect on the polysaccharide structure. Overall, under microwave drying (MD), especially MD-400, the contents of most of the four monosaccharides and oligosaccharides were relatively high, suggesting the structural complexity and bioactivity diversity of the Trichosanthes kirilowii polysaccharides treated by this drying method.

[0054] To further evaluate the differences in Trichosanthes kirilowii polysaccharides treated by different drying methods, a polysaccharide depolymerization fingerprinting method combined with multivariate statistical analysis was used to classify Trichosanthes kirilowii polysaccharide samples treated by different drying methods (see...). Figure 9 ).

[0055] First, principal component analysis was used to classify the polysaccharides from Trichosanthes kirilowii treated by different drying methods. Figure 9 (A). The results show that the variance contribution rates of the first two principal components are 66% and 18.9%, respectively, with a cumulative variance contribution rate of 84.9%. As can be seen from the figure, the air-dried (SD) and oven-dried (HD-80) Trichosanthes kirilowii samples can be well distinguished from Trichosanthes kirilowii samples treated by other drying methods. The two microwave-dried (MD-400 and MD-600) Trichosanthes kirilowii samples are located close to each other.

[0056] Then, the supervised PLS-DA method was used to distinguish the Trichosanthes kirilowii polysaccharide samples treated by different drying methods. Figure 9 (B)(R) 2 X=0.982, R 2 Y=0.954, Q 2 =0.917). The results show that this method can effectively distinguish Trichosanthes kirilowii polysaccharides treated by different drying methods (overall classification accuracy was 100%). The intercept R0 of the 200th permutation test... 2 = 0.146, Q 2= -0.514 Figure 9 The results of the cross-validation (R2= 0.999, RMSE = 0.000, Fig. 6C) and the external validation (R2= 0.999, RMSE = 0.000, Fig. 6D) of the model showed that the model was effective and reliable. In addition, the VIP analysis (VIP > 1, Fig. 6E) showed that peak 11 (unknown), peak 10 (unknown), peak 1 (unknown), peak 8 (glucose), peak 15 (DP2) and peak 5 (galactose) had a greater contribution to the discrimination and could be used as markers for the discrimination of drying methods. Figure 9 D) showed that peak 11 (unknown), peak 10 (unknown), peak 1 (unknown), peak 8 (glucose), peak 15 (DP2) and peak 5 (galactose) had a greater contribution to the discrimination and could be used as markers for the discrimination of drying methods.

[0057] In the present application, a method for determining the depolymerized polysaccharides of Trichosanthes fruit by ultrasonic-assisted acid hydrolysis-HILIC-CAD was established, and 6 monosaccharides, 1 aldehyde acid and 2 oligosaccharides were identified by HPLC-ESI-Q-TOF / MS. The correct discrimination of different medicinal parts (whole Trichosanthes fruit, Trichosanthes fruit peel, Trichosanthes fruit seed and Trichosanthes fruit core) of Trichosanthes fruit was achieved by using the polysaccharide depolymerate fingerprint combined with principal component analysis. The discrimination of Trichosanthes fruit polysaccharides treated by different drying methods was achieved by using the polysaccharide depolymerate fingerprint combined with PLS-DA analysis, and peak 11 (unknown), peak 10 (unknown), peak 1 (unknown), peak 8 (glucose), peak 15 (DP2) and peak 5 (galactose) could be used as markers for discrimination, which provided a method and technical support for the analysis and quality control of Trichosanthes fruit polysaccharides.

[0058] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for constructing a fingerprint spectrum of Trichosanthes kirilowii polysaccharides, characterized in that, Includes the following steps: (1) After drying, the Trichosanthes kirilowii seeds are crushed and sieved to obtain Trichosanthes kirilowii powder; (2) Add deionized water to Trichosanthes kirilowii powder, extract by ultrasonication, filter and collect the filtrate, precipitate with alcohol, and centrifuge to collect the precipitate, which is Trichosanthes kirilowii polysaccharide; (3) After redissolving the Trichosanthes polysaccharide, add trifluoroacetic acid for ultrasonic-assisted acid hydrolysis, dry with nitrogen, and redissolve again in a mixture of acetonitrile and water to obtain the test solution; (4) The test solution was injected into the hydrophilic interaction chromatography-electrospray detector-electrospray-quadrupole-time-of-flight mass spectrometer (HILIC-CAD / ESI-Q-TOF / MS) for detection to obtain 16 chemical components; (5) A fingerprint spectrum of Trichosanthes kirilowii polysaccharide was established using 16 chemical components as common peaks.

2. The construction method as described in claim 1, characterized in that, The drying methods include: air drying, oven drying, freeze drying, and microwave drying, with microwave drying being preferred. Preferably, the drying temperature is 50~90℃; Preferably, the freeze-drying method includes: pre-freezing in a refrigerator at -18~25 ℃ for 10~14 h, then placing it in a vacuum freeze dryer and freeze-drying it to constant weight under the conditions of -35~-45 ℃ in a cold trap and a vacuum degree of 0.8~1.2 Pa. Preferably, the microwave drying conditions are 300~700 W for 15~30 min; Alternatively, Trichosanthes kirilowii includes whole Trichosanthes kirilowii, Trichosanthes kirilowii peel, Trichosanthes kirilowii pulp and Trichosanthes kirilowii seeds, preferably whole Trichosanthes kirilowii or Trichosanthes kirilowii peel, more preferably Trichosanthes kirilowii peel; Alternatively, the dried Trichosanthes kirilowii can be pulverized and sieved through a 50-80 mesh sieve, preferably through a 60 mesh sieve.

3. The construction method as described in claim 1, characterized in that, In step (2), the mass ratio of Trichosanthes kirilowii powder to deionized water is 1:(8~12), preferably 1:10; Alternatively, in step (2), the temperature of ultrasonic extraction is 85~100 ℃, preferably 90 ℃; the power of ultrasonic extraction is 300~500 W, preferably 350 W; and the time of ultrasonic extraction is 20~30 min, preferably 25 min.

4. The construction method as described in claim 1, characterized in that, In step (2), the temperature of alcohol precipitation is 0~6 ℃, preferably 4 ℃; the time of alcohol precipitation is 10~14 h, preferably 12 h; Alternatively, in step (3), the conditions for reconstitution include: the solvent is deionized water; the reconstitution temperature is 80~100 ℃.

5. The construction method as described in claim 1, characterized in that, In step (3), the acid hydrolysis temperature is 70~100 ℃, preferably 80 ℃; the acid hydrolysis time is 10~30 min, preferably 15 min; the ultrasonic power during acid hydrolysis is 240~360 W, preferably 280 W; and the concentration of trifluoroacetic acid during acid hydrolysis is 3~6 mol / L, preferably 4 mol / L. Alternatively, in step (3), the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is (2.5~4):1, preferably 3:

1.

6. The construction method as described in claim 1, characterized in that, In step (4), the chromatographic conditions are as follows: Chromatographic column: Waters Xbridge Amide column; mobile phase A was acetonitrile, mobile phase B was 0.8% formic acid in water, gradient elution; Preferably, the gradient elution conditions include: 0~12 min, 92%~85% A; 12~30 min, 85%~84% A; 30~40 min, 84%~75% A; 40~60 min, 75%~50% A; Preferably, the flow rate during detection is 0.7~1 mL / min, more preferably 0.8 mL / min; Preferably, the injection volume during detection is 18~22 μL, more preferably 20 μL; Preferably, during detection, the column temperature of the chromatographic column is 35~45 ℃, more preferably 40 ℃.

7. The construction method as described in claim 1, characterized in that, In step (4), the detection parameters of the electro-fog detector include: The gas source was N2; the pressure was 8.8 kPa; the filter time was 2.0 sec; and the atomizer temperature was 60 °C.

8. The construction method as described in claim 1, characterized in that, In step (4), the mass spectrometry conditions are: The instrument was used in both positive and negative ionization modes; the operating parameters were as follows: nebulizer pressure was 200 kPa, and drying gas flow rate was 10.0 L·min. -1 The drying gas temperature was 350 °C, the pyrolysis voltage was 10 eV, the capillary voltage for positive and negative ion modes was 3.5 kV and 3.0 kV, respectively, and the scanning range was 50~2000 m / z.

9. The construction method as described in claim 1, characterized in that, In step (4), the 16 chemical components are peak1, peak2, peak3, peak4, peak5, peak6, peak7, peak8, peak9, peak10, peak11 and peak12; The retention times for peak 1 were 7.3–7.7 min; peak 2, 8.8–9.2 min; peak 3, 9.6–10.0 min; peak 4, 11.0–11.4 min; peak 5, 13.8–14.2 min; peak 6, 17.5–17.7 min; peak 7, 19.1–19.5 min; peak 8, 19.8–20.2 min; peak 9, 21.1–21.5 min; and peak 10, 26.9–27.3 min. The retention times for peak 11 are 40.4–40.8 min; peak 12 is 48.8–49.2 min; peak 13 is 51.0–51.4 min; peak 14 is 52.4–52.8 min; peak 15 is 55.1–55.5 min; and peak 16 is 58.0–58.4 min.

10. The application of the Trichosanthes kirilowii polysaccharide fingerprint constructed by the construction method according to any one of claims 1 to 9 in quality evaluation and control.

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