Evaluation method for steaming processing quality of harvested trichosanthes kirilowii maxim

Abstract

The invention provides a method for evaluating steaming processing quality of harvested trichosanthes kirilowii maxim. Comprising the following steps: determining the steaming time of harvested trichosanthes kirilowii maxim by taking the content of trichosanthes kirilowii maxim polysaccharide in a processed trichosanthes kirilowii maxim product, the molecular weight of the trichosanthes kirilowii maxim polysaccharide, the monosaccharide composition of the trichosanthes kirilowii maxim polysaccharide, the chromatographic peak area of a trichosanthes kirilowii maxim polysaccharide depolymer and the pharmacological activity of the processed trichosanthes kirilowii maxim product as evaluation indexes. The invention establishes a method for evaluating the steaming process of the trichosanthes kirilowii maxim by integrating multi-dimensional chemical characterization and pharmacological activity, and the method has important significance for improving the steaming processing quality of the harvested trichosanthes kirilowii maxim.

Description

Technical Field

[0001] This invention relates to the field of medicinal material processing technology, specifically to a method for evaluating the quality of post-harvest steaming processing of Trichosanthes kirilowii. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Trichosanthes kirilowii Maxim. or Trichosanthes rosthornii Harms, both belonging to the Cucurbitaceae family, are dried, ripe fruits. Their pericarp, seeds, and roots are used medicinally as trichosanthes peel, seeds, and root powder, respectively. According to research, trichosanthes was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), listed as a medium-grade herb, and is a commonly used medicine in traditional Chinese medicine for clearing heat and detoxifying. Modern medical research shows that trichosanthes extract has various effects, including dilating coronary arteries, increasing blood flow, improving hypoxia tolerance, lowering serum cholesterol, antibacterial properties, and anticancer effects.

[0004] Post-harvest drying is a crucial step in the production of traditional Chinese medicinal herbs to ensure their efficacy. It inhibits microbial growth, reduces enzyme activity, and slows down many water-mediated reactions. Currently, the traditional method for drying Trichosanthes kirilowii (a type of gourd) involves cutting the fruit along with its stem when it is still green around the autumnal equinox and placing it in a well-ventilated, shaded area to dry. However, this traditional method is inefficient, highly susceptible to weather conditions, and prone to mold and rot, failing to guarantee product quality and yield. The steaming method, which processes immature Trichosanthes kirilowii fruit into "Trichosanthes kirilowii slices," allows moisture to evaporate quickly from the tissue and kills insect eggs, preventing insect infestation. This method not only effectively avoids mold and rot caused by insufficient drying after harvest but also avoids the smoke and dust pollution problems associated with direct baking.

[0005] Furthermore, polysaccharides are composed of more than 10 monosaccharide molecules polymerized through glycosidic bonds. They have large molecular weights, generally tens of thousands or even millions, and are one of the four basic substances constituting life. Polysaccharides are one of the most representative bioactive components of Trichosanthes kirilowii, possessing various pharmacological activities such as antioxidant, anticoagulant, hypoglycemic, antitumor, immunomodulatory, and hepatoprotective effects.

[0006] During the steaming process of Trichosanthes kirilowii fruit, polysaccharides may be lost, affecting the quality of the fruit. Therefore, it is necessary to evaluate the steaming process of Trichosanthes kirilowii to ensure the quality of the steamed fruit. Summary of the Invention

[0007] To overcome the above problems, the present invention provides a method for evaluating the quality of post-harvest steaming processing of Trichosanthes kirilowii.

[0008] To achieve the above technical objectives, the present invention adopts the following technical solution: This invention provides a method for evaluating the quality of post-harvest steaming and processing of Trichosanthes kirilowii, comprising the following steps: The content, molecular weight, composition, chromatographic peak area of ​​depolymerized trichosanthes polysaccharides, and pharmacological activity of the processed trichosanthes product were used as evaluation indicators to determine the postharvest steaming time of trichosanthes.

[0009] In one or more embodiments, the method for post-harvest steaming and processing of Trichosanthes kirilowii includes the following steps: Place the whole Trichosanthes kirilowii fruit in a steamer and steam it using an electromagnetic heating device. After steaming, remove the fruit, flatten it, cut it into strips, and dry it in a forced-air drying oven.

[0010] Preferably, the power of the electromagnetic heating device is 2000~2200 W, and more preferably 2100 W; Preferably, the steaming time is 20-60 minutes, and more preferably 40 minutes.

[0011] Preferably, the drying temperature is 38~42 ℃, more preferably 40 ℃; the drying time is 20~30 h, more preferably 24 h.

[0012] In one or more embodiments, the preparation method of whole Trichosanthes kirilowii polysaccharide includes the following steps: After harvesting, the whole Trichosanthes kirilowii fruit is steamed, dried, pulverized, and sieved to obtain Trichosanthes kirilowii powder. Deionized water is added to the Trichosanthes kirilowii powder, and ultrasonic extraction is performed. The filtrate is collected by filtration, precipitated with alcohol, and the precipitate is collected by centrifugation, which is the whole Trichosanthes kirilowii polysaccharide.

[0013] Preferably, the pulverized material passes through a 50-80 mesh sieve, and more preferably through a 60 mesh sieve.

[0014] Preferably, the mass ratio of Trichosanthes kirilowii powder to deionized water is 1:(8~12), and more preferably 1:10.

[0015] Preferably, the ultrasonic extraction temperature is 85~100 ℃, more preferably 90 ℃; the ultrasonic extraction power is 300~500w, more preferably 350w; and the ultrasonic extraction time is 20~30 min, more preferably 25 min.

[0016] Preferably, the temperature for alcohol precipitation is 0~6 ℃, more preferably 4 ℃; the time for alcohol precipitation is 10~14 h, more preferably 12 h.

[0017] In one or more embodiments, the content of whole Trichosanthes kirilowii polysaccharides is determined by the phenol-sulfuric acid method.

[0018] In one or more embodiments, the molecular weight of Trichosanthes kirilowii polysaccharide was determined using the HPGPC-ELSD method.

[0019] In one or more embodiments, the composition of Trichosanthes kirilowii polysaccharide is determined by ion chromatography.

[0020] Preferably, the detection method includes: reconstitute the whole Trichosanthes polysaccharide, add trifluoroacetic acid for ultrasonic-assisted acid hydrolysis, dry the acid hydrolysis product with nitrogen and wash it repeatedly with methanol, reconstitute it with water and then determine it by ion chromatography.

[0021] More preferably, the resolution conditions include: the solvent is deionized water; and the resolution temperature is 80~100 ℃.

[0022] More preferably, the acid hydrolysis temperature is 80~100 ℃, more preferably 90 ℃; the acid hydrolysis time is 1.5~3 h, more preferably 2 h; and the concentration of trifluoroacetic acid during acid hydrolysis is 3~6 mol / L, more preferably 4 mol / L.

[0023] More preferably, the chromatographic conditions include: column: Dionex™ CarboPac™ PA 20; mobile phase A is H2O, and mobile phase B is 20 mmol·L⁻¹. -1 NaOH solution, mobile phase C is 20 mmol·L⁻¹ -1 NaOH and 200 mmol·L - 1 NaOAc mixed solution, mobile phase D is 200 mmol·L⁻¹ -1 NaOH solution, linear gradient elution.

[0024] More preferably, the elution conditions include: 0~20 min, 10 mmol·L -1 NaOH; 20.1~40 min, 62 mmol·L -1 NaOH- 100 mmol·L -1 NaOAc; 40.1~50 min, 20 mmol·L -1 NaOH- 200 mmol·L -1 NaOAc.

[0025] More preferably, the injection volume is 25 µL and the flow rate is 0.4 mL·min. -1 The column temperature is 30 °C.

[0026] In one or more embodiments, the preparation method of the whole Trichosanthes kirilowii polysaccharide depolymer includes the following steps: The whole Trichosanthes polysaccharide was reconstituted and then subjected to acid hydrolysis with trifluoroacetic acid, followed by drying to obtain the depolymerized whole Trichosanthes polysaccharide.

[0027] Preferably, the resolution conditions include: the solvent is deionized water; and the resolution temperature is 80~100 ℃.

[0028] Preferably, the acid hydrolysis temperature is 70~100 ℃, more preferably 80 ℃; the acid hydrolysis time is 4.5~6 h, more preferably 5 h; and the concentration of trifluoroacetic acid during acid hydrolysis is 3~6 mol / L, more preferably 4 mol / L.

[0029] Preferably, the method for detecting the chromatographic peak area of ​​the whole Trichosanthes kirilowii polysaccharide depolymerization product includes the following steps: The depolymerized whole Trichosanthes kirilowii polysaccharide was redissolved in a mixed solution of acetonitrile and water to obtain the test solution; The test solution was injected into a hydrophilic interaction chromatography-electro-atomization detector (HILIC-CAD) for detection, and 16 chemical components were obtained; the peak areas of chromatographic peaks 5, 8, 15 and 16 were detected.

[0030] More preferably, the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is (2.5~4):1, more preferably 3:1.

[0031] More preferably, 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.

[0032] More 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.

[0033] More preferably, during detection, the flow rate is 0.7~1 mL / min, preferably 0.8 mL / min; the injection volume is 18~22 μL, preferably 20 μL; and the column temperature is 35~45 ℃, preferably 40 ℃.

[0034] More preferably, 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.

[0035] More preferably, the retention time of chromatographic peak 5 is 13.8~14.2 min; the retention time of chromatographic peak 8 is 19.8~20.2 min; the retention time of chromatographic peak 15 is 55.1~55.5 min; and the retention time of chromatographic peak 16 is 58.0~58.4 min.

[0036] In one or more embodiments, the pharmacological activity includes in vitro antioxidant activity, in vitro hypoglycemic activity, and in vitro anticoagulant activity.

[0037] Preferably, the in vitro antioxidant activity includes hydroxyl radical scavenging activity and ABTS scavenging activity.

[0038] Preferably, the in vitro hypoglycemic activity includes α-amylase inhibitory activity and α-glucosidase inhibitory activity.

[0039] Preferably, the in vitro anticoagulant activity is thrombin inhibitory activity.

[0040] The beneficial effects of this invention are as follows: This invention provides a method for evaluating the quality of postharvest steaming processing of Trichosanthes kirilowii, comprising the following steps: using the content, molecular weight, composition, chromatographic peak area of ​​the depolymerized polysaccharide, and pharmacological activity of the processed whole Trichosanthes kirilowii product as evaluation indicators to determine the steaming time. Studies show that with increasing steaming time, the content of whole Trichosanthes kirilowii polysaccharide first increases and then decreases; the molecular weight of whole Trichosanthes kirilowii polysaccharide first increases and then decreases; the monosaccharide composition also changes significantly; the chromatographic peak area of ​​the depolymerized polysaccharide shows an overall trend of first decreasing, then increasing, and then decreasing again with increasing time; the profile of the depolymerized polysaccharide combined with PCA analysis can accurately distinguish Trichosanthes kirilowii samples with different steaming times. Experiments on the in vitro antioxidant activity (hydroxyl radical and ABTS), anticoagulant activity, and hypoglycemic ability (α-amylase, α-glucosidase) of Trichosanthes kirilowii polysaccharide under different steaming times showed that the activity generally increased and then decreased with increasing steaming time. Specifically, after steaming for 40 min, the Trichosanthes kirilowii polysaccharide exhibited relatively high content, large molecular weight, and good pharmacological activity. This suggests that short-term steaming can promote the release, polymerization, and increase the molecular weight of Trichosanthes kirilowii polysaccharide, thus enhancing its pharmacological activity; however, long-term steaming leads to depolymerization, decreased content and molecular weight, and consequently, reduced pharmacological activity. This invention establishes a method integrating multidimensional chemical characterization and pharmacological activity to evaluate the steaming process of Trichosanthes kirilowii, which is of great significance for improving the quality of postharvest steaming processing of Trichosanthes kirilowii. Attached Figure Description

[0041] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0042] Figure 1 The graph shows the changes in the content (a) and molecular weight (HPGPC-ELSD) of whole Trichosanthes kirilowii polysaccharides under different steaming times; Figure 2 A diagram showing the monosaccharide composition of different parts of Trichosanthes kirilowii under different steaming times; Figure 3 HILIC-CAD plots (a), characteristic peak distribution (b), and PCA analysis (c) of Trichosanthes kirilowii polysaccharides under different steaming times. Detailed Implementation

[0043] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0044] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0045] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0046] The Trichosanthes kirilowii used in the following examples were harvested from the Pingyin planting base of Jinan Hebao Chinese Medicinal Materials Co., Ltd., and identified by Qilu University of Technology (Shandong Academy of Sciences) as the mature fruit of Trichosanthes kirilowii Maxim, a plant of the Cucurbitaceae family.

[0047] Example 1 1.1 Sample processing and preparation of whole Trichosanthes kirilowii polysaccharide: (1) Sample preparation: Take Trichosanthes kirilowii samples of similar size, label them, randomly group them, place them in a steamer, and steam them in an induction cooker (power of 2100 W) for 20, 30, 40, 50 and 60 min respectively. After steaming, take them out, flatten them, cut them into strips, and dry them in an electric hot air drying oven at 40 °C for 24 h. Crush the dried whole Trichosanthes kirilowii into powder and pass it through a 60 mesh sieve for later use.

[0048] (2) Extraction of whole Trichosanthes kirilowii polysaccharide: Accurately weigh 1.0 g of whole Trichosanthes kirilowii powder and place it in a stoppered Erlenmeyer flask. Add 10 mL of 90 °C deionized water and extract for 25 min in an ultrasonic extractor (350 W). Filter the supernatant and add 95% ethanol to the filtrate. Place the flask in a refrigerator at 4 °C for ethanol precipitation for 12 h. Centrifuge again (4500 g) for 15 min. Evaporate the precipitate to dryness in an 80 °C water bath and redissolve it with 5 mL of hot water. Transfer the precipitate to a 10 mL volumetric flask and add water to make up to volume.

[0049] 1.2 Determination of whole Trichosanthes kirilowii polysaccharide content: Preparation of standard curve: Accurately pipette glucose standard solution (0.1 mg / mL) -1 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1 mL, 2 mL, and 4 mL were respectively added to 10 mL volumetric flasks, and diluted to volume with water. Then, 1 mL of each flask was added to a dry test tube, and 1.0 mL of 5% phenol solution was added to each tube. The tubes were then shaken well, and 5 mL of sulfuric acid solution was added along the wall. The tubes were shaken for 6 min, ensuring the gas was released by opening the lid. After standing for 5 min, the tubes were heated in a boiling water bath for 10 min. After cooling to room temperature, the absorbance was measured at 486 nm using a 1 cm cuvette. A standard curve was plotted with concentration on the x-axis and absorbance on the y-axis. The regression equation was y = 25.611x + 0.092, R0. 2 = 0.9943. The results indicate that glucose solution 0~0.02 mg·mL -1 It exhibits a good linear relationship with absorbance.

[0050] 1.3 Determination of molecular weight of Trichosanthes kirilowii polysaccharide: The relative molecular mass of Trichosanthes kirilowii polysaccharide was determined by HPGPC-ELSD method. An Agilent 1260 high-performance liquid chromatograph was used, with an Ultrahydrogel™ column (7.8 × 300 mm, 10 μm), a mobile phase of 10 mmol ammonium acetate solution, an injection volume of 20 μL, a column temperature of 30 °C, and a flow rate of 0.5 mL / min. -1 The ELSD gas flow rate is 1.6 L·min. -1 The drift tube temperature was 80 °C. HPGPC-ELSD chromatograms of polysaccharide standards with different relative molecular masses (1, 5, 10, 20, 40, 70 kDa) were established. A linear fit was performed with the logarithm of the standard molecular weight as the ordinate and the retention time of the corresponding chromatographic peak as the abscissa, yielding the standard curve equation: lgMw = -0.4583t + 10.197 (R²). 2 =0.9701).

[0051] 1.4 Determination of monosaccharide composition: (1) Chromatographic conditions: Thermo ICS 5000 ion chromatography system (ICS 5000, Thermo Fisher Scientific, USA); detector: electrochemical detector; column: Dionex™ CarboPac™ PA 20 (150×3.0 mm, 10 μm) column; injection volume: 25 µL; mobile phase A was H2O, mobile phase B was 20 mmol·L⁻¹ -1 NaOH solution, mobile phase C is 20 mmol·L⁻¹ -1 NaOH and 200 mmol·L -1 NaOAc mixed solution, mobile phase D is 200 mmol·L⁻¹ -1 NaOH solution, linear elution. Flow rate 0.4 mL / min. -1 Column temperature: 30 °C; Elution program: 0–20 min, 10 mmol·L⁻¹ - 1 NaOH; 20.1~40 min, 62 mmol·L -1 NaOH- 100 mmol·L -1 NaOAc; 40.1~50 min, 20 mmol·L - 1 NaOH- 200 mmol·L -1 NaOAc.

[0052] (2) Preparation of reference standards and test samples: Reference solution: Accurately weigh 1 mg of anhydrous glucose standard into a 10 mL volumetric flask, dilute to volume with water to prepare a 0.1 mg / mL solution. -1 A standard solution of 1 mg / mL was prepared. -1 Reference solutions: rhamnose, arabinose, galactose, glucose, xylose, fructose, galacturonic acid, and glucuronic acid. These solutions were serially diluted with water to concentrations of 100, 50, 10, 5, 1, 0.5, and 0.1 μg / mL. -1 The monosaccharide reference standard mixture was stored at 4 °C for later use.

[0053] Test solution: Pipette 700 μL of the whole Trichosanthes kirilowii polysaccharide solution from step 1.1 into a hydrolysis tube, and add 4.0 mol·L⁻¹ -1 A 700 μL solution of trifluoroacetic acid (TFA) was prepared, sealed, and then sonicated (90 °C, 300 W) for 2 h. The acid hydrolysis product was dried under nitrogen and washed repeatedly with methanol to remove TFA residue. It was then reconstituted with 1 mL of water, filtered through a 0.22 µm filter membrane, and stored at 4 °C for later use.

[0054] 1.5 HPLC-CAD Analysis of the Depolymerized Polysaccharides from Trichosanthes kirilowii Polysaccharide depolymerization conditions: Take 700 µL of whole Trichosanthes kirilowii polysaccharide solution from step 1.1 and add an appropriate amount of 4 mol·L⁻¹ polysaccharide. -1 The trifluoroacetic acid solution was hydrolyzed at 80 °C for 5 h, dried under nitrogen, and then reconstituted with 1 mL of solvent (acetonitrile:water = 1:1). After passing through a 0.22 µm filter membrane, the solution was stored at 4 °C for later use.

[0055] Chromatographic conditions: Waters Xbridge Amide column (4.6 mm × 250 mm, 5 μm, Waters Corporation, USA); mobile phase A: acetonitrile; mobile phase B: 0.8% formic acid in water. Gradient elution (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. Charged Aerosol Detection (CAD) parameters: gas source: N₂; pressure: 8.8 kPa; filter time: 2.0 sec; nebulizer temperature: 60°C.

[0056] 1.6 Experimental methods for different pharmacological activities (1) In vitro antioxidant scavenging experiment: Hydroxyl radical scavenging assay: In vitro hydroxyl radical scavenging experiments were conducted on whole Trichosanthes kirilowii treated with different methods. 50 µL of FeSO4 (3 mmol·L⁻¹) was added sequentially to each well of a 96-well plate. -1 50 µL of salicylic acid-ethanol solution (6 mmol·L⁻¹), 50 µL of whole Trichosanthes kirilowii polysaccharide solution. Add 50 µL of H₂O₂ (3 mmol·L⁻¹). -1 The reaction was started and proceeded for 30 minutes in the dark, protected from light. The absorbance (A) was measured at 510 nm using a microplate reader. Methanol served as the blank control, and ascorbic acid (Vc) served as the positive control. The reaction was repeated three times. The hydroxyl radical scavenging rate was calculated using the formula.

[0057] Clearance rate (%) = ×100; (1) In the formula A 样品 A 空白 The values ​​are A for the sample group and the blank group, respectively.

[0058] ABTS Scavenging Assay: In vitro ABTS scavenging experiments were conducted on whole Trichosanthes kirilowii treated with different methods. ABTS working solution was prepared by reacting ABTS solution (7.0 mM, H2O) and potassium persulfate (4.9 mM, H2O) at 4 °C in the dark for 12 h. A series of control solutions of various concentrations were prepared. 50 μL of the test solution and 100 μL of the working solution were added to 96-well plates, and the absorbance (A) was measured at 734 nm using a microplate reader after 6 min in the dark. Methanol served as the blank control, and ascorbic acid (Vc) served as the positive control. The experiment was repeated three times. The ABTS free radical scavenging rate was calculated using the formula.

[0059] Clearance rate (%) = ×100; (2) In the formula A 样品 A 空白 The values ​​are A for the sample group and the blank group, respectively.

[0060] (2) In vitro hypoglycemic activity assay: α-Amylase Activity Inhibition Detection: In vitro α-amylase activity scavenging experiments were conducted on whole Trichosanthes kirilowii treated with different methods. 100 μL of sample extract or standard was mixed with α-amylase solution (100 μL, 2 U·mL⁻¹). -1 Dissolves in 0.1 mol·L⁻¹ -1 Mix with pH 6.8 PBS buffer and incubate at 37 °C for 10 min. Then add 200 μL of substrate-soluble starch solution (1 mg / mL). -1 The mixture was stirred at 37 °C for 20 min, and the reaction was terminated by adding 200 μL of 3,5-dinitrosalicylic acid reagent (DNS) solution. Finally, the mixture was heated in boiling water for 10 minutes, cooled, and the absorbance at 540 nm was measured using a microplate reader. No sample was added to the blank group, no α-amylase solution was added to the background group, and acarbose served as the positive control. The α-amylase inhibitory activity was calculated as follows: Clearance rate (%) = ×100; (3) In the formula A 样品 A 空白 A 背景 The values ​​are A for the sample group and the blank group, respectively.

[0061] α-Glucosidase Activity Inhibition Detection: In vitro α-glucosidase activity scavenging experiments were conducted on whole Trichosanthes kirilowii treated with different methods. 100 μL of sample extract or standard was mixed with α-glucosidase solution (40 μL, 0.5 U·mL⁻¹). -1 Dissolves in 0.1 mol·L⁻¹ -1Mix with pH 6.8 PBS buffer and incubate at 37 °C for 30 min. Then add 100 μL of substrate PNPG (4 mmol·L⁻¹). -1 Dissolved in 0.1 M pH 6.8 PBS buffer and incubated at 37 °C for 10 min, then add 50 μL of 0.1 mol·L⁻¹ PBS. -1 The reaction was terminated with Na2CO3 solution, and the absorbance at 405 nm was measured using a microplate reader. No sample was added to the blank group, no α-amylase solution was added to the background group, and acarbose served as the positive control. The α-glucosidase inhibitory activity was calculated as follows: Clearance rate (%) = ×100; (4) In the formula A 样品 A 空白 A 背景 The values ​​are A for the sample group and the blank group, respectively.

[0062] (3) In vitro anti-inflammatory activity assay: The anti-inflammatory activity of whole Trichosanthes kirilowii treated with different methods was determined. In a 96-well plate, 100 μL of test solution and 75 μL of thrombin solution (1.75 U·mL⁻¹) were added. -1 Incubate at room temperature for 15 min. After incubation, add 25 μL of 150 M chromogenic substrate simultaneously using a multichannel pipette to begin detection. Measure absorbance at 405 nm every 20 seconds for 15 minutes. All experiments were performed three times, and the inhibition rate was the average of the three observations. The blank control was 0.5% DMSO phosphate buffer (75 mM, pH 7.4). The positive control was argatroban. Thrombin inhibitory activity was calculated as follows: Clearance rate (%) = ×100; (5) In the formula A 样品 A 空白 A 背景 The values ​​are A for the sample group and the blank group, respectively.

[0063] Example 2 (1) Effect of steaming time on the polysaccharide content of whole Trichosanthes kirilowii: The polysaccharide content of Trichosanthes kirilowii and its steamed products was determined by the phenol-sulfuric acid method (see...). Figure 1(a) The experiment found that the total sugar content of Trichosanthes kirilowii changed significantly after steaming compared with that of unsteamed Trichosanthes kirilowii. The polysaccharide content of whole Trichosanthes kirilowii showed a trend of first increasing and then decreasing with increasing steaming time, especially with the highest content at 40 min, reaching 27.46%; followed by 50 min and 30 min, with contents of 26.53% and 26.07%, respectively. This is because short-term steaming causes hydrolysis of cellulose, pectin, etc. in Trichosanthes kirilowii, producing water-soluble polysaccharides with low degree of polymerization, thus increasing the polysaccharide content; however, with increasing steaming time, the polysaccharides are affected by high-temperature steam, causing glycosidic bond breakage and generating monosaccharides or oligosaccharides, thus causing the polysaccharide content to show a decreasing trend again.

[0064] (2) Effect of steaming time on the molecular weight of Trichosanthes kirilowii polysaccharide: By establishing HPGPC-ELSD chromatograms of polysaccharide standards with different relative molecular masses, and using the logarithm of the standard molecular weight as the ordinate and the retention time of the corresponding chromatographic peak as the abscissa, a linear fit was performed, yielding the standard curve equation: lgMw = -0.4583t + 10.197(R²). 2 = 0.9701). HPGPC-ELSD plots of whole Trichosanthes kirilowii polysaccharides at different steaming times ( Figure 1 (b) Both contained two chromatographic peaks (a and b), indicating that the whole Trichosanthes kirilowii polysaccharide mainly contains polysaccharides with two molecular weight ranges. The molecular weight changes over time are shown in Table 1. Table 1 shows that the relative molecular masses of whole Trichosanthes kirilowii polysaccharide a and b initially increased and then decreased with time. The relative molecular masses of a and b in whole Trichosanthes kirilowii reached their maximum values ​​after 50 min of steaming, being 11.70 ± 0.04 kDa and 6.08 ± 0.02 kDa, respectively. It is speculated that polymerization occurred in the whole Trichosanthes kirilowii polysaccharide during the initial stage of steaming, resulting in an increase in molecular weight; with increasing steaming time, the prolonged high temperature caused the glycosidic bonds to break, reducing the molecular weight of the polysaccharide.

[0065] Table 1. Relative molecular mass of peaks under different distillation methods

[0066] (3) Effect of steaming time on the monosaccharide composition of Trichosanthes kirilowii polysaccharide The monosaccharide composition of the whole Trichosanthes kirilowii polysaccharide was determined by ion chromatography, such as... Figure 2As shown, the whole Trichosanthes kirilowii polysaccharide is composed of glucose (Glc), arabinose (Ara), galactose (Gal), rhamnose (Rha), fructose (Fru), xylose (Xyl), glucuronic acid (GlcA), and galacturonic acid (GalA). Among them, glucose, arabinose, and galactose are the most abundant in the whole Trichosanthes kirilowii polysaccharide at different steaming times. Comparison of different steaming stages revealed significant differences in monosaccharide composition and content: the highest monosaccharide content was found at 0 min, followed by 50 min and 40 min. This is because there is a certain relationship between monosaccharide composition and polysaccharide content and structure. This is because short-term high-temperature steaming may promote the degradation of cellulose and pectin, producing polysaccharides with low polymerization degree; in addition, short-term high temperature may also cause a certain amount of whole Trichosanthes kirilowii polysaccharide to polymerize, both of which increase the total amount of whole Trichosanthes kirilowii polysaccharide, thus increasing the measured monosaccharide composition. However, as steaming time increases, high temperatures may cause the glycosidic bonds in polysaccharides to break, leading to a decrease in polysaccharide content and consequently a decrease in monosaccharide composition. In summary, polysaccharides with different monosaccharide compositions may be selectively retained or degraded during steaming due to differences in solubility or thermal stability, ultimately affecting the results of monosaccharide composition detection.

[0067] (4) Effect of steaming time on the depolymerization of Trichosanthes kirilowii polysaccharide The changes in the chromatograms of the depolymerized whole Trichosanthes kirilowii polysaccharide under different steaming times were investigated using the HILIC-CAD method. The HILIC-CAD chromatograms of whole Trichosanthes kirilowii polysaccharide under different steaming times are shown below. Figure 3 Figure a shows that the distribution of the fingerprint peaks of the whole Trichosanthes kirilowii polysaccharide changed with increasing steaming time. To further investigate the changes of the whole Trichosanthes kirilowii polysaccharide during the steaming process, several representative chromatographic peaks (peaks 5, 8, 15, and 16) were selected to study the effect of different steaming times on the fingerprint of the whole Trichosanthes kirilowii polysaccharide. Figure 3 (b) In the whole Trichosanthes kirilowii fruit, except for chromatographic peak 16, the areas of the other three chromatographic peaks generally showed a trend of first decreasing, then increasing, and then decreasing again with time. Among them, the peak areas of each chromatographic peak reached their maximum values ​​at 40 min. Chromatographic peak 16 had the highest content in the un-steamed form, and its peak area was closely followed at 40 min. Overall, the fingerprint chromatograms of whole Trichosanthes kirilowii polysaccharides differed under different steaming times, with the largest peak areas at 40 min of steaming.

[0068] Sixteen major chromatographic peaks in the whole Trichosanthes kirilowii sample were designated as "common peaks." Similarity analysis of the fingerprint chromatograms of whole Trichosanthes kirilowii polysaccharides at different steaming times was performed using the correlation coefficient method. The results are shown in Table 2. The table shows that the fingerprint of whole Trichosanthes kirilowii polysaccharides initially increased and then decreased with increasing steaming time. This indicates that the structure of the whole Trichosanthes kirilowii polysaccharides changed during steaming, necessitating further analysis using other techniques.

[0069] Table 2. Similarity analysis of fingerprint chromatograms of Trichosanthes kirilowii polysaccharides under different steaming times.

[0070] Multivariate statistical analysis was used to classify whole Trichosanthes kirilowii polysaccharide samples to characterize the effect of steaming time on whole Trichosanthes kirilowii polysaccharides. Principal component analysis was employed to analyze the fingerprints of whole Trichosanthes kirilowii polysaccharides with different steaming times (see [link to study]). Figure 3 (c) The first two principal components (PC1 and PC2) contributed 52.1% and 18.8% of the distillation difference, respectively. Figure 3 As shown in Figure c, the HILIC-CAD-based fingerprinting of whole Trichosanthes kirilowii polysaccharides combined with PCA analysis can distinguish Trichosanthes kirilowii samples with different steaming times. In particular, the fingerprint of whole Trichosanthes kirilowii polysaccharides steamed for 40 min shows a clear distinction from those steamed at other times. These results indicate that the structure of whole Trichosanthes kirilowii polysaccharides changes during steaming, and the fingerprinting of whole Trichosanthes kirilowii polysaccharide depolymerization combined with multivariate statistical analysis can be used to distinguish Trichosanthes kirilowii samples based on steaming time.

[0071] (5) Effect of steaming time on the pharmacological activity of Trichosanthes kirilowii polysaccharide: In vitro antioxidant activity: The inhibition rates of antioxidant activity (OH•, ABTS) of whole Trichosanthes kirilowii polysaccharide under different steaming times were calculated according to formulas (1, 2), as shown in Tables 3 and 4. The tables show that within the range of 0–60 min, the antioxidant activity of whole Trichosanthes kirilowii polysaccharide initially increased and then decreased with increasing steaming time. This is presumably due to changes in the molecular weight of the polysaccharide depolymerization caused by prolonged high temperatures. The antioxidant activity (OH•, ABTS) of whole Trichosanthes kirilowii polysaccharide was highest after steaming for 40 min, followed by steaming for 30 min and 50 min. However, overall, the antioxidant activity of whole Trichosanthes kirilowii polysaccharide after steaming was higher than that of unsteamed Trichosanthes kirilowii.

[0072] Table 3. Hydroxyl radical scavenging activity (%) of Trichosanthes kirilowii polysaccharides before and after steaming

[0073] Table 4. ABTS scavenging activity of Trichosanthes kirilowii polysaccharides before and after steaming (%)

[0074] In vitro hypoglycemic activity: The hypoglycemic activity (α-amylase, α-glucosidase) inhibition rates of whole Trichosanthes kirilowii polysaccharides under different steaming times were calculated according to formulas (3, 4), as shown in Tables 5 and 6. In the α-amylase inhibition experiment, the activity of whole Trichosanthes kirilowii fractions was highest after steaming for 40 min, followed by 50 min and 30 min. In the α-glucosidase inhibition experiment, the activity of whole Trichosanthes kirilowii polysaccharides was highest after steaming for 40 min, followed by 30 min and 50 min. Overall, steaming time significantly affected the hypoglycemic activity of whole Trichosanthes kirilowii polysaccharides, with the highest activity at 40 min. At this time, the α-amylase inhibitory activity of whole Trichosanthes kirilowii polysaccharides was close to that of the positive control acarbose at the same concentration (0.6 mg·mL⁻¹). However, longer steaming times led to changes in the molecular weight of the polysaccharides due to depolymerization, resulting in decreased activity.

[0075] Table 5. α-Amylase inhibitory activity of Trichosanthes kirilowii polysaccharides before and after steaming (%)

[0076] Table 6. α-glucosidase inhibitory activity of Trichosanthes kirilowii polysaccharides before and after steaming (%)

[0077] In vitro anticoagulant activity The anticoagulant activity of Trichosanthes kirilowii polysaccharides from different parts of the plant was calculated according to formula (5), as shown in Table 7. The thrombin inhibitory activity of whole Trichosanthes kirilowii polysaccharides showed a trend of first increasing and then decreasing with increasing steaming time. Among them, the highest activity was observed after steaming for 40 min, followed by steaming for 30 min and steaming for 50 min. It can be seen that steaming time also affects the anticoagulant activity of whole Trichosanthes kirilowii polysaccharides.

[0078] In summary, steaming time significantly affects the antioxidant, hypoglycemic, and anticoagulant activities of Trichosanthes kirilowii polysaccharides. Based on changes in polysaccharide content and molecular weight, monosaccharide composition, and polysaccharide depolymerization profiles during steaming, it can be inferred that short-term steaming promotes the release, polymerization, and increase in molecular weight of Trichosanthes kirilowii polysaccharides, resulting in enhanced activity; however, prolonged steaming leads to depolymerization, decreased content and molecular weight of Trichosanthes kirilowii polysaccharides, and a corresponding reduction in activity.

[0079] Table 7. Thrombin activity (%) of different parts of Trichosanthes kirilowii before and after steaming

[0080] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for evaluating the quality of post-harvest steaming and processing of Trichosanthes kirilowii, characterized in that, Includes the following steps: The content, molecular weight, composition, chromatographic peak area of ​​depolymerized trichosanthes polysaccharides, and pharmacological activity of the processed trichosanthes product were used as evaluation indicators to determine the postharvest steaming time of trichosanthes.

2. The evaluation method as described in claim 1, characterized in that, The post-harvest steaming and processing method of Trichosanthes kirilowii includes the following steps: Place the whole Trichosanthes kirilowii fruit in a steamer and steam it using an electromagnetic heating device. After steaming, remove the fruit, flatten it, cut it into strips, and dry it in a forced-air drying oven. Preferably, the power of the electromagnetic heating device is 2000~2200 W, and more preferably 2100 W; Preferably, the steaming time is 20-60 minutes, and more preferably 40 minutes; Preferably, the drying temperature is 38~42 ℃, more preferably 40 ℃; the drying time is 20~30 h, more preferably 24 h.

3. The evaluation method as described in claim 1, characterized in that, The preparation method of Trichosanthes kirilowii polysaccharide includes the following steps: After harvesting, the whole Trichosanthes kirilowii fruit is steamed, dried, pulverized, and sieved to obtain Trichosanthes kirilowii powder. Deionized water is added to the Trichosanthes kirilowii powder, and ultrasonic extraction is performed. The filtrate is collected by filtration, precipitated with alcohol, and the precipitate is collected by centrifugation, which is the whole Trichosanthes kirilowii polysaccharide. Preferably, the pulverized material passes through a 50-80 mesh sieve, and more preferably through a 60 mesh sieve; Preferably, the mass ratio of Trichosanthes kirilowii powder to deionized water is 1:(8~12), more preferably 1:10; Preferably, the ultrasonic extraction temperature is 85~100 ℃, more preferably 90 ℃; the ultrasonic extraction power is 300~500 W, more preferably 350 W; and the ultrasonic extraction time is 20~30 min, more preferably 25 min. Preferably, the temperature for alcohol precipitation is 0~6 ℃, more preferably 4 ℃; the time for alcohol precipitation is 10~14 h, more preferably 12 h.

4. The evaluation method as described in claim 1, characterized in that, The content of polysaccharides in whole Trichosanthes kirilowii was determined by the phenol-sulfuric acid method; Alternatively, the molecular weight of Trichosanthes kirilowii polysaccharide was determined using the HPGPC-ELSD method.

5. The evaluation method as described in claim 1, characterized in that, The composition of the whole Trichosanthes kirilowii polysaccharide was determined by ion chromatography; Preferably, the detection method includes: reconstitute the whole Trichosanthes polysaccharide, add trifluoroacetic acid for ultrasonic-assisted acid hydrolysis, dry the acid hydrolysis product with nitrogen and wash it repeatedly with methanol, reconstitute it with water, and then determine it by ion chromatography.

6. The evaluation method as described in claim 5, characterized in that, The conditions for redissolution include: the solvent is deionized water; the redissolution temperature is 80~100 ℃; Alternatively, the acid hydrolysis temperature is 80~100 ℃, preferably 90 ℃; the acid hydrolysis time is 1.5~3 h, preferably 2 h; and the concentration of trifluoroacetic acid during acid hydrolysis is 3~6 mol / L, preferably 4 mol / L. Alternatively, the chromatographic conditions include: column: Dionex™ CarboPac™ PA 20; mobile phase A: H₂O, mobile phase B: 20 ​​mmol·L⁻¹ -1 NaOH solution, mobile phase C is 20 mmol·L⁻¹ -1 NaOH and 200 mmol·L -1 NaOAc mixed solution, mobile phase D is 200 mmol·L⁻¹ -1 NaOH solution, linear gradient elution; Preferably, the elution conditions include: 0~20 min, 10 mmol·L -1 NaOH; 20.1~40 min,62 mmol·L -1 NaOH-100 mmol·L -1 NaOAc; 40.1~50 min,20 mmol·L -1 NaOH-200 mmol·L -1 NaOAc; Preferably, the injection volume is 25 µL and the flow rate is 0.4 mL·min. -1 The column temperature is 30 °C.

7. The evaluation method as described in claim 1, characterized in that, The preparation method of the whole Trichosanthes kirilowii polysaccharide depolymer includes the following steps: The whole Trichosanthes polysaccharide was reconstituted and then acid-hydrolyzed with trifluoroacetic acid, and dried to obtain the depolymerized whole Trichosanthes polysaccharide; Preferably, the resolution conditions include: the solvent is deionized water; the resolution temperature is 80~100 ℃; Preferably, the acid hydrolysis temperature is 70~100 ℃, more preferably 80 ℃; the acid hydrolysis time is 4.5~6 h, more preferably 5 h; and the concentration of trifluoroacetic acid during acid hydrolysis is 3~6 mol / L, more preferably 4 mol / L.

8. The evaluation method as described in claim 1, characterized in that, The method for detecting the chromatographic peak area of ​​the depolymerized Trichosanthes kirilowii polysaccharide includes the following steps: The depolymerized whole Trichosanthes kirilowii polysaccharide was redissolved in a mixed solution of acetonitrile and water to obtain the test solution; The test solution was injected into a hydrophilic interaction chromatography-electro-fogging detector (HILIC-CAD) for detection, and 16 chemical components were obtained; the peak areas of chromatographic peaks 5, 8, 15 and 16 were detected. Preferably, the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is (2.5~4):1, more preferably 3:

1.

9. The evaluation method as described in claim 8, characterized in that, 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, during detection, the flow rate is 0.7~1 mL / min, more preferably 0.8 mL / min; the injection volume is 18~22 μL, more preferably 20 μL; and the column temperature is 35~45 ℃, more preferably 40 ℃. Preferably, 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. Preferably, the retention time of chromatographic peak 5 is 23.8-14.2 min; the retention time of chromatographic peak 8 is 19.8-20.2 min; the retention time of chromatographic peak 15 is 55.1-55.5 min; and the retention time of chromatographic peak 16 is 58.0-58.4 min.

10. The evaluation method as described in claim 1, characterized in that, The pharmacological activities include in vitro antioxidant activity, in vitro hypoglycemic activity, and in vitro anticoagulant activity; Preferably, the in vitro antioxidant activity includes hydroxyl radical scavenging activity and ABTS inhibitory activity; Preferably, the in vitro hypoglycemic activity includes α-amylase inhibitory activity and α-glucosidase inhibitory activity; Preferably, the in vitro anticoagulant activity is thrombin activity.

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