Method for purifying total phenylpropanoid from lonicera macranthoides extract and application of total phenylpropanoid

Abstract

The invention relates to a method for purifying total phenylpropanoid from a lonicera macranthoides extract and application of the total phenylpropanoid in medicines. Relates to the technical field of traditional Chinese medicines, in particular to application of lonicera macranthoides total phenylpropanoid (TP) in preparation of medicines with functions of promoting oxidation resistance, reducing blood sugar, inhibiting cholinesterase and resisting inflammation. According to the present invention, a lonicera macranthoides extract is purified by using a macroporous resin, in particular an HPD100 resin, to obtain high-purity TP. The method comprises the following specific steps: (1) loading a sample containing the lonicera macranthoides extract into a macroporous adsorption resin column with the diameter-height ratio of 1: 5 at the flow rate of 1.5-2.5 mL / min; (2) eluting by using deionized water to remove unadsorbed liquid, wherein the dosage is 40-120mL; and (3) carrying out elution by using a 60% alcoholic solution at an elution flow rate of 1.5-2.5 mL / min to obtain the lonicera macranthoides TP. In-vitro experiments show that the purified product has remarkable antioxidant activity, blood sugar lowering activity, acetylcholin esterase inhibition activity and anti-inflammatory activity.

Description

Technical Field

[0001] The present invention relates to a method for purifying total phenylpropanoids from the extract of Lonicera macranthoides Hand.-Mazz. and its application in drugs, belonging to the technical field of traditional Chinese medicine. Background Art

[0002] Lonicera macranthoides Hand.-Mazz. is a plant of the genus Lonicera in the family Caprifoliaceae. Its dried flower buds or newly opened flowers are used as medicine and are one of the source plants of the traditional Chinese medicine Flos Lonicerae Confusae. It can also be used as food and is a traditional Chinese medicine with the property of "both medicine and food". This plant has the effects of clearing heat and detoxifying, and dispersing wind-heat, and is mainly used to treat diseases such as wind-heat cold and febrile diseases caused by warm pathogens. Modern research shows that Lonicera macranthoides Hand.-Mazz. has antioxidant, antibacterial, anti-inflammatory and immunomodulatory properties and has potential health and economic value. Its functional components include bioactive substances such as phenylpropanoids, triterpenoid saponins and iridoids. Among them, phenylpropanoid compounds are the main active components of Lonicera macranthoides Hand.-Mazz. and have diverse biological activities. The main phenylpropanoid compounds in Lonicera macranthoides Hand.-Mazz. include chlorogenic acid (CA), isochlorogenic acid A (IAA), isochlorogenic acid C (IAC) and neochlorogenic acid (NA), which have significant antioxidant, anti-inflammatory and antibacterial and other pharmacological activities.

[0003] Chinese invention patent CN108743629A discloses a preparation method of total phenylpropanoid components in Lepidogrammitis drymoglossoides (Baker) Ching. The preparation method is as follows: (1) crushing the Lepidogrammitis drymoglossoides (Baker) Ching medicinal materials and screening them through a 40-mesh sieve, and drying them at 55-65 °C; (2) soaking the dried medicinal materials with an ethanol solution with a volume fraction of 65-75%, then performing ultrasonic extraction, extracting 2-4 times, 40-50 minutes each time, filtering and combining the extraction solutions, and concentrating under reduced pressure to obtain an extract; (3) dissolving the extract with an appropriate amount of water, first passing it through a macroporous adsorption resin column, and sequentially eluting with water and ethanol with different concentrations (20%, 40%, 60%, 70%, 80%, 95%) in a gradient manner, and collecting the 70% ethanol elution part; (4) separating the collected 70% ethanol elution part by silica gel column chromatography, and eluting with chloroform-methanol with a volume ratio of 90:1, 75:1, 45:1, 26:1, 14:1, 5:1 in sequence, collecting the 14:1 elution part and concentrating to obtain the phenylpropanoid components in Lepidogrammitis drymoglossoides (Baker) Ching. However, this preparation method uses a large amount of organic solvents such as chloroform, which is toxic to a certain extent, and the operation is complex and requires high.

[0004] At present, there are few reports on the purification and application of TP in Lonicera macranthoides Hand.-Mazz. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to purify total phenylpropanoids from the extract of Lonicera macranthoides Hand.-Mazz. to achieve significant antioxidant, hypoglycemic, acetylcholinesterase inhibitory and anti-inflammatory activity effects.

[0006] To solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A method for purifying total phenylpropanoids from the extract of Lonicera macranthoides Hand.-Mazz., comprising the following steps: Step S1: Loading a sample solution containing the extract of Lonicera macranthoides Hand.-Mazz. (LME) at a flow rate of 1.5 - 2.5 mL / min onto a macroporous adsorption resin column with a resin bed diameter-to-height ratio of 1:5 for adsorption; the sample solution is an aqueous solution of the extract of Lonicera macranthoides Hand.-Mazz., with a concentration of 2.5 - 12.5 mg / mL; the sample loading amount of the sample solution is 300 mL;

[0008] Step S2: Removing impurities, using deionized water to remove impurities, with the flow rate of impurity removal elution being 1.5 - 2.5 mL / min, and controlling the amount of deionized water used for impurity removal elution to be 40 - 120 mL;

[0009] Step S3: Eluting, using an alcohol solution with a volume concentration of 60% for elution, controlling the elution flow rate to be 1.5 - 2.5 mL / min, collecting the eluate to obtain Lonicera macranthoides Hand.-Mazz. TP.

[0010] The preparation method of the extract of Lonicera macranthoides Hand.-Mazz. includes the steps: pulverizing Lonicera macranthoides Hand.-Mazz. and passing it through an 80-mesh sieve, adding 60% methanol in a ratio of 1:10 (g:mL), performing ultrasonic extraction for 40 min at a power of 400 W, twice in total, filtering, concentrating the filtrate under reduced pressure at 40 - 60 °C, and drying under reduced pressure to obtain the extract of Lonicera macranthoides Hand.-Mazz..

[0011] In step S1, the macroporous adsorption resin is selected from one of the macroporous resins HPD-100, AB-8, SP825, D 101, HPD-600, S-8, DM130, HPD-450, NAK-9.

[0012] In step S2, deionized water is used to remove impurities.

[0013] The amount of the deionized water used is 40 - 120 mL.

[0014] In step S3, the elution flow rate is 1.5 - 2.5 mL / min.

[0015] The application of purifying total phenylpropanoids from the extract of Lonicera macranthoides Hand.-Mazz. is for preparing drugs with antioxidant, hypoglycemic, acetylcholinesterase inhibitory and anti-inflammatory activities.

[0016] The beneficial effects of adopting the above technical solutions are:

[0017] The present invention evaluated nine macroporous resins for the purification of TP from LME. HPLC analysis determined that the most effective TP purification resin was HPD100. Through systematic optimization, the optimal adsorption and desorption conditions were determined: LME concentration 10 mg / mL, sample solution pH 3, resin dosage 7 g, eluent concentration 60%, eluent volume 30 mL, sample loading flow rate 1.5 mL / min, total sample loading volume 200 mL, elution flow rate 2.0 mL / min, and total eluent volume 220 mL. Kinetic studies showed that the adsorption process conforms to the pseudo-second-order kinetic model, while the equilibrium data best fits the Temkin isotherm model. Thermodynamic analysis indicated that the adsorption process is spontaneous and exothermic. In addition, molecular docking studies showed a strong binding affinity between the resin and the target compound. In vitro bioactivity assays confirmed that both LME and LMEP have significant antioxidant, hypoglycemic, AChE inhibitory, and anti-inflammatory activity effects. Description of the Drawings

[0018] Figure 1 For the screening of different types of macroporous resins.

[0019] Figure 2a For the effect of pH in static adsorption / desorption experiments.

[0020] Figure 2b For the effect of sample concentration in static adsorption / desorption experiments.

[0021] Figure 2c For the effect of resin dosage in static adsorption / desorption experiments.

[0022] Figure 2d For the effect of eluent concentration in static adsorption / desorption experiments.

[0023] Figure 2e For the effect of eluent volume in static adsorption / desorption experiments.

[0024] Figure 3 For dynamic adsorption / desorption.

[0025] In the figure: A) Investigation of different sample loading flow rates, B) Investigation of different elution flow rates.

[0026] Figure 4 For the antioxidant activities of LME and LMEP.

[0027] In the figure: A) DPPH radical scavenging activity, B) ABTS radical scavenging activity.

[0028] Figure 5 For the enzyme inhibitory activities of LME and LMEP.

[0029] In the figure: A) α-Glucosidase inhibitory activity, B) α-Amylase inhibitory activity, C) Acetylcholinesterase inhibitory activity.

[0030] Figure 6 Effect on cell viability

[0031] In the figure: A) Effect of LMEP on the viability of Raw264.7 cells, B) Effect of LMEP on the viability of LPS-induced Raw264.7 cells, C) Effect of LMEP on the levels of IL-1β and IL-16 in LPS-induced Raw264.7 cells. Compared with the control, ##P<0.01, P<0.001, compared with LPS, ***P<0.001. Detailed implementation mode

[0032] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0033] 1 Materials and methods

[0034] 1.1 Materials and reagents

[0035] Lonicera macranthoides Hand.-Mazz., HPD-100, AB-8, SP825, D101, HPD-600, S-8, DM130, HPD-450, NAK-9 resins, anhydrous methanol, anhydrous ethanol, hydrochloric acid, chromatographic methanol, chromatographic acetonitrile, ABTS, DPPH, PNPG, DTNB, potassium persulfate, sodium carbonate, AChE, acetylthiocholine iodide (ATCI), acarbose, vitamin C (Vc), α-glucosidase (α-Glu), α-amylase.

[0036] 1.2 Main instrument and equipment

[0037] High performance liquid chromatograph, constant temperature oscillator, ultrasonic cleaner, rotary evaporator, vacuum drying oven, multi-functional pulverizer, ultraviolet-visible spectrophotometer, microplate reader.

[0038] 1.3 Test methods

[0039] 1.3.1 Determination of TP content

[0040] The content of TP was determined by high performance liquid chromatography (HPLC). Standard samples of NA, CA, IAA, and IAC were accurately weighed, dissolved and diluted to a series of concentration standard solutions, and analyzed after passing through a membrane. For chromatographic analysis, a Phenomenex C18 chromatographic column (250×4.6 mm, 5 μm) was used, the flow rate was 0.6 mL / min, the column temperature was 30 °C, and the injection volume was 10 μL. An EClas s i cal 3200 chromatograph was used for analysis, equipped with an ECl as s i cal D3200 series ultraviolet-visible detector and an ECl as s i cal O3200 series column oven. The detection wavelength was set at 320 nm, and the mobile phase gradient elution conditions were as follows: 0 min, 10% acetonitrile; 5 min, 20% acetonitrile; 10 min, 40% acetonitrile; 30 min, 65% acetonitrile; 35 min, 10% acetonitrile. The aqueous phase was 0.2% formic acid water. A linear regression model was established with the concentration as the abscissa (x-axis) and the peak area as the ordinate (y-axis).

[0041] 1.3.2 Preparation of LME

[0042] Lonicera macranthoides Hand.-Mazz. was crushed with a multi-functional crusher and passed through an 80-mesh sieve. The sieved powder was mixed with 60% methanol at a ratio of 1:10 (g / mL), ultrasonic extraction was carried out at a power of 400 W for 40 min, and the extraction was carried out twice in total. The filtrates were combined; the filtrate was concentrated by a rotary evaporator, dissolved in anhydrous methanol and dried in a vacuum drying oven until no liquid remained, and LME was obtained.

[0043] 1.3.3 Pretreatment of macroporous resin

[0044] The pretreatment of the macroporous resin was carried out according to the improved method

[27] . Nine different types of macroporous resins were respectively immersed in anhydrous ethanol for more than 12 h, and repeatedly washed with distilled water until no ethanol residue remained and then reserved.

[0045] 1.3.4 Screening of different types of macroporous resins

[0046] 1.0 g of each different type of macroporous resin was taken and placed in a 50 mL conical flask respectively, 25 mL of TPE (5 mg / mL) was added, the conical flask was sealed and shaken in a constant temperature oscillator for 24 h (37 °C, 150 rpm), the resin and the solution were separated by vacuum filtration, the content of TP in the filtrate was measured by HPLC and the adsorption rate and adsorption capacity were calculated; for each macroporous resin saturated with adsorption, 25 mL of anhydrous methanol was added respectively, shaken for 24 h under the same conditions, the content of TP in the filtrate was measured after filtration, and the desorption rate and desorption capacity were calculated.

[0047] Adsorption rate (%) = [(C0×V1 - C e ×V2) / C0×V1]×100

[0048] Adsorption capacity (mg / g) = [(C0 × V1 – C e × V2)] / W

[0049] Desorption rate (%) = (C d × V d ) / [(C0 × V1 – C e × V2)] × 100

[0050] Desorption amount (mg / g) = (C d × V d ) / W C0, C e , C d (mg / mL) represent the concentrations of TP in the initial solution, equilibrium solution, and desorbing solution respectively; V1, V2, V d (mL) are the sample volume, equilibrium volume, and eluent volume in sequence; W (g) represents the amount of macroporous resin used.

[0051] 1.3.5 Static adsorption and desorption experiments

[0052] Precisely weigh a certain amount of macroporous resin and mix it with 25 mL of LME solution. Investigate the effects of different mass concentrations (2.5 - 12.5 mg / mL), pH values (2 - 6), resin dosages (1 - 9 g), eluent concentrations (20 - 100%), and eluent volumes (20 - 40 mL) on adsorption and desorption, and optimize the purification conditions.

[0053] 1.3.6 Dynamic adsorption and desorption experiments

[0054] Fill a certain volume of macroporous resin into a chromatography column, and set the column volume to 20 mL (1 BV). Rinse the resin column with deionized water until the eluent is clear and there is no ethanol residue. Control the sample loading rate (1.5 - 2.5 mL / min) through a constant flow pump. After the sample completely passes through the resin column, rinse the column with deionized water to remove the residual liquid between the resins. Subsequently, adjust the eluent flow rate (1.5 - 2.5 mL / min) through a constant flow pump for elution. During the adsorption and desorption processes, collect the eluate in 20 - mL fractions per tube, and collect a total of 15 tubes. Determine the TP content in the eluate by high - performance liquid chromatography (HPLC) and calculate its concentration.

[0055] 1.3.7 Biological activity investigation

[0056] 1.3.7.1 DPPH free radical scavenging experiment

[0057] According to

[28] and with slight adjustment, TPE and TPEP were dissolved and sample solutions with different concentration gradients (0.1–0.5 mg / mL) were prepared. The sample solutions were mixed with 0.2 mmol / L DPPH solution at a volume ratio of 1:2, reacted in the dark at room temperature for 30 min, and the absorbance was measured at 517 nm. The solvent without the sample was used as the blank, and Vc was used as the positive control to quantify the free radical scavenging activity.

[0058] DPPH scavenging rate (%) = [(A b - A s ) / A b × 100%

[0059] A b and A s are the absorbance values of the blank group and the sample group, respectively.

[0060] 1.3.7.2 ABTS free radical scavenging experiment

[0061] With reference to

[29] and with slight modification, 7 mmol / L ABTS solution and 5 mmol / L potassium persulfate solution were mixed in equal volumes and placed in the dark for at least 16 hours to prepare the ABTS free radical stock solution. Before use, the stock solution was diluted to an absorbance of 0.70 ± 0.02 (λ = 734 nm). LME and LMEP (0.04–0.08 mg / mL) reacted with the working solution at a ratio of 1:3 (v / v) in the dark for 7 minutes. The absorbance was measured at 734 nm, and the scavenging rate was calculated by the following formula, with VC as the positive control.

[0062] ABTS scavenging rate (%) = (A b - A s ) / A b × 100%

[0063] A b and A s represent the absorbance values of the blank group and the sample group, respectively.

[0064] 1.3.7.3 α-Glu inhibition experiment

[0065] According to existing research

[30] with minor adjustments, the experiment was divided into an experimental group: 80 μL PBS (pH 6.8, 0.1 M) + 20 μL sample + 25 μL α-Glu (0.2 U / mL), a blank group: 105 μL PBS + 20 μL sample, a negative control group: 100 μL PBS + 25 μL α-Glu. After reacting at 37 °C for 20 min, 25 μL PNPG (4 mM) was added, and after continuing to react for 15 min, 50 μL Na2CO3 (0.2 M) was added to terminate the reaction. The absorbance was measured at 405 nm and the enzyme inhibition rate was calculated. Acarbose was used as a positive control, and the reaction was carried out in a 96-well plate.

[0066] Inhibition rate (%) = [1 - (A s - A b ) / A n × 100%

[0067] A b 、A s and A n represent the absorbance values of the blank group, experimental group, and negative control group, respectively. 1.3.7.4 α-Amylase inhibition experiment

[0068] According to previous research

[31] with slight adjustments, 500 μL of the sample was mixed with 500 μL of α-amylase (0.1 mg / mL), reacted at 37 °C for 10 min, and after adding 500 μL of starch (1%), the reaction continued for 10 min. Subsequently, 1 mL of DNS was added, boiled for 5 min, cooled, and diluted with 4 mL of deionized water. The absorbance A s was measured at 520 nm. The absorbance A b was measured with PBS (pH 6.8, 0.1 M) replacing α-amylase, and the absorbance A n was measured with PBS replacing the sample, and the enzyme inhibition rate was calculated according to the following formula, with acarbose as the positive control.

[0069] Inhibition rate (%) = [A n - (A s - A b )] / A n × 100%

[0070] 1.3.7.5 AChE inhibition experiment

[0071] Referring to existing research

[32] and making modifications, the experimental group A1 was set up: 130 μL PBS (pH

[0072] 8.0 - 9.5, 0.1M) + 20 μL AChE (1.6 mg / mL) + 30 μL sample + 20 μL ATCI (15 mM), blank group A2: 180 μL PBS + 20 μL AChE, control group A3: 160 μL PBS + 20 μL AChE + 20 μL ATCI, background group A4: 150 μL PBS + 20 μL AChE + 30 μL sample. React at 37 °C for 30 min, add 20 μL DTNB and let stand at room temperature for 30 min. Measure the absorbance at 405 nm. The reaction is carried out in a 96-well plate, with tacrine as the positive control.

[0073] Inhibition rate (%) = [(A3 - A2) - (A1 - A2)] / (A3 - A2) × 100%

[0074] 1.3.8 Cell viability assay

[0075] Purchase Raw264.7 cells from Wuhan Pricella Biotechnology Co., Ltd. and culture them in DMEM medium containing 10% (v / v) fetal bovine serum and 1% (v / v) antibiotics. Use the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric method [33, 34] to detect cell viability. Collect cells with 80% confluence and inoculate them into a 96-well plate at a density of 3×10 4 cells per well. After incubation for 24 hours, treat the cells with different concentrations of LMEP (0, 25, 50, 100, 200, 400 μg / mL) to determine the optimal concentration. Meanwhile, another group of cells is inoculated under the same conditions. After incubation for 24 hours, co-treat the cells with 10 μg / mL lipopolysaccharide (LPS) and different concentrations of LMEP (25, 50, 100, 200, 400 μg / mL) for 24 hours to evaluate the effect of LMEP on the cytotoxicity of LPS-induced Raw264.7 cells. Subsequently, add 10 μL (5 mg / mL) of MTT solution to each well and continue incubation for 4 hours. After removing the supernatant, add 150 μL of dimethyl sulfoxide (DMSO) to all wells and incubate at room temperature for 10 minutes, then measure the absorbance (OD) value at 570 nm. Calculate the cell viability through the following formula:

[0076] Cell survival rate (%) = OD 样品 / OD 空白 × 100%

[0077] where OD 样品 represents the absorbance of the sample group, and OD 空白 represents the absorbance of the blank group.

[0078] 1.3.9 Determination of Inflammatory Factors

[0079] Cells in the logarithmic growth phase were seeded into 96-well plates at a density of 3×10 4 cells per well and cultured in an incubator for 24 hours. Subsequently, they were co-treated with different concentrations (100 μg / mL and 400 μg / mL) of LMEP and 50 μL of LPS (1 μg / mL). After 24 hours of culture, the supernatant was collected, and the concentrations of IL-16 and IL-1β were measured using an ELISA kit according to the standard experimental procedure.

[0080] 1.3.10 Data Processing

[0081] Data are expressed as mean ± standard deviation. One-way analysis of variance (one-way ANOVA) was used between different groups, and P < 0.05 indicates statistical significance.

[0082] 2 Results and Discussion

[0083] 2.1 Screening of Different Types of Macroporous Resins Due to the differences in the structures and properties of phenylpropanoid compounds, the adsorption and desorption abilities of macroporous resins for them also vary. Different resins have unique chemical and physical properties, which significantly affect their adsorption and desorption behaviors towards target components

[35] . The selection of resins mainly depends on key parameters such as polarity, specific surface area, and pore size [36, 37]. Figure 1 The adsorption and desorption rates of nine different types of macroporous resins for TP are shown. Among them, SP825 resin shows the highest adsorption capacity for LME, but its desorption efficiency is poor; while HPD600 resin, although its adsorption performance is relatively weak, exhibits the best desorption ability; the purification efficiencies of AB-8 and HPD 100 resins are similar. Phenylpropanoid compounds in Lonicera macranthoides Hand.-Mazz. are highly polar and are more easily adsorbed by resins with higher polarity

[38] , but the pore size and specific surface area of the resin also have a significant impact on the adsorption performance. Considering the comprehensive performance and cost-effectiveness, HPD 100 macroporous resin was finally selected for the subsequent experiments.

[0084] Table 1 Physicochemical Properties of Different Types of Resins

[0085]

[0086] 2.2 Investigation of Sample pH

[0087] The pH value of the sample solution has a crucial impact on the adsorption capacity of macroporous resin. The phenylpropanoid compounds in Lonicera macranthoides Hand.-Mazz. are polyhydroxyphenolic acids

[39] , which mainly exist in molecular form under acidic conditions. This molecular state can enhance their affinity with macroporous resin, thereby increasing the adsorption capacity

[40] . Experiments show that when the pH value rises from 2 to 3, the adsorption rate of HPD 100 macroporous resin gradually increases; while when the pH value exceeds 3, its adsorption capacity begins to decline sharply. At pH 3, the resin achieves the best adsorption effect, as compared with the adsorption rates under other pH conditions, such as Figure 2a shown. When the pH exceeds 3, the carboxyl and hydroxyl groups within the molecule gradually undergo deprotonation and exist in ionic form

[41] , resulting in a decrease in their adsorption affinity with the resin. Given that there is no significant difference in the adsorption capacity between pH 2 and pH 3 conditions, pH 3 was finally selected as the optimal pH value of the sample solution.

[0088] 2.3 Investigation of sample concentration

[0089] The adsorption capacity of macroporous resin for the target compound is affected by the mass concentration of the sample solution, and the results are as shown in Figure 2b shown. The adsorption capacity gradually increases with the increase in concentration and reaches a peak when the concentration reaches 10 mg / mL; when the sample concentration is further increased beyond this concentration, the adsorption capacity decreases significantly. At low concentrations, the adsorption capacity is positively correlated with the LME level, which is due to the increased utilization rate of the binding active sites of the target compound; while beyond this threshold, the increase in LME will promote the competitive adsorption of impurities on the resin surface

[22] , resulting in a slight decrease in the adsorption efficiency. Therefore, the loading concentration of HPD 100 was finally selected as 10 mg / mL.

[0090] 2.4 Investigation of solid-liquid ratio

[0091] As shown in Figure 2c shown, it shows the effect of resin dosage on the adsorption of the target compound. As the resin dosage increases, the adsorption efficiency of the target compound gradually increases. When the resin dosage reaches 7 g, further increasing the dosage does not result in a significant improvement in the adsorption efficiency. Considering the resin utilization rate and cost, 7 g was finally selected as the optimal resin dosage.

[0092] 2.5 Investigation of desorbent concentration

[0093] As shown in Figure 2d shown, the desorption capacity of the target compound increases with the increase in the volume fraction of the eluent and reaches a maximum when the volume fraction reaches 60%. Further increasing the volume fraction results in a decrease in the desorption rate. According to the experimental results, the desorbent concentration was finally optimized to 60% for subsequent experiments

[0094] 2.6 Investigation of desorbent volume

[0095] The influence of the volume of the eluent on the desorption ability is as follows Figure 2e shown. When the volume of the eluent increased from 20 mL to 30 mL, the desorption rate of the resin increased and reached the maximum value at 30 mL; when the volume of the eluent continued to increase after exceeding this volume, the desorption efficiency of the resin did not change significantly. Therefore, 30 mL was finally determined as the elution volume.

[0096] The object of the present invention is to provide a preparation method of phenylpropanoid components in Lonicera macranthoides Hand.-Mazz. At the same time, further study the antioxidant, hypoglycemic and other activities of TP in Lonicera macranthoides Hand.-Mazz., and broaden its huge application potential in drug applications.

[0097] 2.7 Leakage curve

[0098] The dynamic leakage curve of HPD 100 was plotted with the volume of the eluate as the abscissa and the concentration of the target compound in the eluate as the ordinate. The volume corresponding to the concentration of the target compound in the eluate reaching 10% of the initial solution concentration was defined as the leakage point

[42] . The earlier the leakage point appears, the lower the adsorption efficiency of the macroporous resin. When the flow rate was 1.5 mL / min, the leakage point appeared after about 200 mL of the solution passed through the S resin column; when the flow rate was increased to 2.0 mL / min, the leakage point was about 180 mL; while when the flow rate was 2.5 mL / min, the leakage point was about 200 mL, as Figure 3 shown in A of. Therefore, the loading flow rate was finally selected as 1.5 mL / min and the loading volume was 200 mL.

[0099] 2.8 Elution curve

[0100] To further optimize the elution flow rate and elution volume, the dynamic elution curves at different flow rates were plotted by monitoring the change of the concentration of the target compound in the eluate with time. As Figure 3 shown in B of, when the flow rate was 1.5 mL / min, about 300 mL of the eluent was required to complete the elution process; when the flow rate was increased to 2.0 mL / min, the required eluent volume was reduced to 220 mL; while when the flow rate was 2.5 mL / min, about 260 mL of the eluent was required for complete elution. Therefore, to maximize the elution efficiency, the optimal elution flow rate was finally selected as 2.0 mL / min, and the corresponding elution volume was set as 260 mL.

[0101] 2.9 Content determination

[0102] The total phenylpropanoid content of Lonicera macranthoides Hand.-Mazz. was expressed by the sum of the contents of chlorogenic acid, neochlorogenic acid, isochlorogenic acid A and isochlorogenic acid C, and linear regression equations were established for quantification respectively. Neochlorogenic acid: y = 32950x – 744.54, R 2 = 0.9996, chlorogenic acid: y = 23411x – 2903.7, R 2= 0.9997, Isochlorogenic acid A: y = 56074x - 1539, R 2 = 0.9992, Isochlorogenic acid C: y = 51638x – 1617.8, R 2 = 0.9995.

[0103] The limits of detection (LODs) and limits of quantification (LOQs) for the four compounds were 0.00836–0.12648 mg / mL and 0.02535–0.38328 mg / mL, respectively. The methodology validation of LME was also carried out. Intra-day precision was determined by six repeated analyses within a single day, and inter-day precision was determined by repeated analyses for six consecutive days. The results showed that the relative standard deviations (RSDs) of intra-day and inter-day precision were both less than 2%, indicating that the analytical method had high precision. The sample stability was investigated at different time points at room temperature, and the RSD values were all less than 2%, confirming the good stability of the four components in the extract. The method repeatability was further verified by parallel analysis of six identical samples, and the obtained RSD values were all less than 2%, indicating good method reproducibility. The recovery rate was determined by the standard addition method, and the recovery rate was calculated after adding known amounts of the four standard products to LME. The results showed that the recovery rates of each component of this method met the requirements.

[0104] 2.10 Activity investigation

[0105] 2.10.1 Antioxidant experiment

[0106] To evaluate the antioxidant activities of LME and LMEP, two different determination methods were adopted. As Figure 4 shown, within the tested concentration range, the scavenging of DPPH and ABTS free radicals by LME and LMEP showed concentration dependence. At the maximum tested concentration, LME and LMEP showed the best scavenging ability for DPPH and ABTS free radicals. At the same time, under the same experimental conditions, the scavenging efficiency of the positive control was greater than 90%.

[0107] The above results indicated that both LME and LMEP showed significant free radical scavenging ability, which demonstrated their potential for development as natural antioxidants.

[0108] 2.10.2 Hypoglycemic experiment

[0109] α-Glu is a membrane-bound enzyme in the small intestine that can catalyze the hydrolysis of α-1,4-glycosidic bonds in oligosaccharides and disaccharides, releasing glucose. Inhibiting α-Glu can slow down the process of complex carbohydrates being broken down into absorbable monosaccharides, thereby reducing the sharp rise in postprandial blood glucose and helping to maintain blood glucose homeostasis

[46] . α-Amylase is secreted by the pancreas and can break down starch into maltose and oligosaccharides, which are further digested into glucose in the small intestine, increasing glucose absorption. Inhibiting α-amylase can slow down starch digestion and reduce glucose bioavailability, thus alleviating postprandial hyperglycemia [31, 47].

[0110] As Figure 5 shown in A and B of , both LME and LMEP samples showed concentration-dependent inhibitory effects on α-Glu and α-amylase, reaching the maximum inhibitory effect at their respective optimal concentrations. The inhibitory rate of the positive control drug acarbose on both enzymes was greater than 90%.

[0111] 2.10.3 AChE Inhibition Experiment

[0112] AChE inhibitors are the main drug class for treating neurodegenerative diseases and act by competitively or non-competitively binding to the active site of the enzyme. This inhibitory effect can prevent the breakdown of acetylcholine, causing it to accumulate in the synaptic region. Enhanced cholinergic signaling can strengthen communication between neurons, thereby alleviating cognitive impairments associated with neurodegenerative diseases (such as Alzheimer's disease)

[48] . As Figure 5 shown in C of , within the test concentration range (0.6–1.0 mg / mL), both LME and LMEP showed significant concentration-dependent AChE inhibitory effects and reached the maximum inhibitory effect at their respective maximum concentrations. The inhibitory rate of the positive control drug tacrine was greater than 70%.

[0113] 2.11 Cell Viability

[0114] The MTT colorimetric method was used to evaluate the effect of LMEP on the viability of Raw264.7 cells, and the results are shown in A of . After treatment with different concentrations of LMEP, the cell viability exceeded 100%. Figure 6 shown in B of , which shows the effect of co-treatment with different concentrations of LMEP and LPS on cell viability. After co-treatment, the cell viability remained above 80%, indicating that LMEP had no cytotoxicity to cells when co-administered with LPS within the test concentration range. Therefore, LMEP concentrations of 100 and 400 μg / mL were selected for subsequent experiments. Figure 6

[0115] 2.15 Inflammatory Factor Assay

[0116] During the inflammatory response, the severity of cellular inflammation can be indirectly reflected by changes in the levels of interleukin IL-16 and IL-1β. Therefore, detecting the levels of IL-16 and IL-1β in Raw264.7 cells helps to understand the anti-inflammatory effect of LMEP. As Figure 6 shown in C and D of

[0117] 3 Conclusions

[0118] In this study, nine macroporous resins were evaluated for the purification of TP from LME. HPLC analysis determined that the most effective TP purification resin was HPD100. Through systematic optimization, the optimal adsorption and desorption conditions were determined: LME concentration 10 mg / mL, sample solution pH 3, resin dosage 7 g, eluent concentration 60%, eluent volume 30 mL, sample loading flow rate 1.5 mL / min, total sample loading volume 200 mL, elution flow rate 2.0 mL / min, total eluent volume 220 mL. Kinetic studies showed that the adsorption process conforms to the pseudo-second-order kinetic model, while the equilibrium data best fit the Temkin isotherm model. Thermodynamic analysis indicated that the adsorption process was spontaneous and exothermic. In addition, molecular docking studies showed a strong binding affinity between the resin and the target compound. In vitro bioactivity assays confirmed that both LME and LMEP had significant antioxidant, hypoglycemic, AChE inhibitory, and anti-inflammatory activities.

Claims

1. A method for purifying total phenylpropanoids from Lonicera macranthoides Hand.-Mazz. extract, characterized in that: It includes the following steps: Step S1: The sample solution containing Lonicera macranthoides extract (LME) is loaded onto a macroporous adsorption resin column with a resin bed diameter-to-height ratio of 1:5 at a flow rate of 1.5 - 2.5 mL / min for adsorption; the sample solution is an aqueous solution of Lonicera macranthoides extract with a concentration of 2.5 - 12.5 mg / mL; the sample volume of the sample solution is 300 mL. Step S2: Impurity removal is carried out using deionized water. The flow rate of impurity removal elution is 1.5 - 2.5 mL / min, and the amount of deionized water used for impurity removal elution is controlled to be 40 - 120 mL. Step S3: Elution is carried out using an alcohol solution with a volume concentration of 60%. The elution flow rate is controlled to be 1.5 - 2.5 mL / min, and the eluate is collected to obtain Lonicera macranthoides TP.

2. The method for purifying total phenylpropanoids from the extract of Lonicera macranthoides Hand.-Mazz. according to claim 1, wherein: The preparation method of the Lonicera macranthoides extract includes the steps: Lonicera macranthoides is crushed and passed through an 80-mesh sieve, 60% methanol is added at a ratio of 1:10 (g:mL), and ultrasonic extraction is carried out at a power of 400 W for 40 min, twice in total. After filtration, the filtrate is concentrated under reduced pressure at 40 - 60 °C and dried under reduced pressure to obtain the Lonicera macranthoides extract.

3. The method for purifying total phenylpropanoids from Lonicera macranthoides Hand.-Mazz. extract according to claim 1, wherein: In step S1, the macroporous adsorption resin is selected from one of the macroporous resins HPD-100, AB-8, SP825, D101, HPD-600, S-8, DM130, HPD-450, NAK-9.

4. The method for purifying total phenylpropanoids from the extract of Lonicera macranthoides Hand.-Mazz. according to claim 1, wherein: In step S2, impurity removal is carried out using deionized water. The amount of the deionized water is 40 - 120 mL.

5. The method for purifying total phenylpropanoids from the extract of Lonicera macranthoides Hand.-Mazz. according to claim 1, characterized in that: In step S3, the elution flow rate is 1.5 - 2.5 mL / min.

6. The application of purifying total phenylpropanoids from the extract of Lonicera macranthoides Hand.-Mazz., as claimed in claim 1, wherein: The application is for the preparation of drugs with antioxidant, hypoglycemic, acetylcholinesterase inhibitory, and anti-inflammatory activities.

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