A method for extracting and purifying total iridoid glycosides from vaccinium uliginosum and application thereof

Abstract

The application discloses a method for extracting and purifying total iridoid glycosides in Lonicera caerulea and application thereof, and comprises the following steps: (1) extraction: a certain amount of Lonicera caerulea powder is weighed, methanol solution is added, and heating is performed to 40-60 DEG C, ultrasonic-assisted extraction is performed for 20-40 min, filtration is performed, methanol is recovered by reduced pressure concentration, and Lonicera caerulea extract is obtained; (2) purification: the Lonicera caerulea extract is subjected to macroporous adsorption resin column chromatography, elution is performed with ethanol, ethanol is recovered by reduced pressure concentration, and total iridoid glycosides are obtained. The application can effectively improve the extraction amount of total iridoid glycosides in Lonicera caerulea and the purity of the extract.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction technology, and in particular to a method for extracting and purifying total iridoid glycosides from honeysuckle fruit and its application. Background Technology

[0002] Honeysuckle berry (Lonicera caerulea L.), also known as blue honeysuckle or haskap, is a plant belonging to the genus Lonicera in the family Caprifoliaceae. It is a newly emerging wild berry resource. According to the "Pharmacopoeia of Changbai Mountain Plants," honeysuckle berry is bitter and cool in nature, and can clear heat and purge fire, dissipate carbuncles and reduce swelling. Modern research shows that honeysuckle berry is rich in various bioactive components, including anthocyanins, polyphenols, flavonoids, and iridoid glycosides. Several iridoid glycosides, such as loganic acid and strychnine, have been identified in its fruit, making it a medicinal and edible resource plant that has attracted much attention in recent years. Honeysuckle berry possesses physiological activities such as scavenging free radicals, inhibiting the phosphorylation of proteins related to inflammatory pathways, and inhibiting cancer cell proliferation. Its antioxidant, lipid-regulating, anti-tumor, and radiation-protective health benefits can be applied to multiple functional food fields, including regulating intestinal flora structure, anti-cancer, anti-obesity, and vision protection.

[0003] Iridoid glycosides are a class of monoterpenoid compounds with a basic cyclopentanepyran ring structure. They commonly exist in plants as glycosides, and pure products are mostly white crystals or powders, readily soluble in polar solvents such as water and methanol. Modern pharmacological studies have found that isolated and identified iridoid glycosides possess a wide range of pharmacological activities, including antiviral, anti-inflammatory, antitumor, hepatoprotective, hypoglycemic, lipid-lowering, and neuroprotective effects. Studies have shown that iridoid glycosides have promising applications in the treatment of diabetes, antitumor activity, immune system regulation, blood system protection, and hepatoprotection. Furthermore, secoiridoid glycosides (such as gentiopicrin, swertiamarin, and swertiamarin) have also been shown to have hepatoprotective, hypoglycemic, lipid-regulating, anti-inflammatory, antitumor, and neuroprotective effects.

[0004] However, current research on the systematic extraction and purification of iridoid glycosides from honeysuckle berries is relatively insufficient. Existing studies mostly focus on anthocyanins or polyphenols, limiting the development and utilization of these components. Current technologies for the extraction and purification of total iridoid glycosides from honeysuckle berries have several shortcomings. Firstly, traditional iridoid glycoside extraction methods often employ solvent extraction (such as water extraction and alcohol extraction), frequently requiring the addition of calcium carbonate or barium hydroxide to the plant material to inhibit enzyme activity and neutralize phytic acids. However, this method suffers from high solvent consumption, long extraction times, and low yields. Furthermore, the glycosidic bonds of iridoid glycosides are easily hydrolyzed to produce unstable aglycones, and improper control of factors such as temperature, pH, and light during extraction can lead to the degradation of active ingredients. These problems not only affect the extraction efficiency and product purity of iridoid glycosides from honeysuckle berries but also restrict their further processing and application in pharmaceuticals, food, and health products. Summary of the Invention

[0005] The purpose of this invention is to provide a method and application for the extraction and purification of total iridoid glycosides from honeysuckle fruit, which can effectively improve the extraction yield and purity of total iridoid glycosides from honeysuckle fruit.

[0006] The technical solution adopted by this invention to solve its technical problem is: A method for extracting and purifying total iridoid glycosides from honeysuckle fruit, comprising the following steps: (1) Extraction: Weigh out honeysuckle berry powder, add methanol solution, heat to 40-60℃, extract with ultrasonic assistance for 20-40 min, filter, concentrate under reduced pressure to recover methanol, and obtain honeysuckle berry extract; (2) Purification: The extract of honeysuckle fruit was subjected to macroporous adsorption resin column chromatography, eluted with ethanol, and concentrated under reduced pressure to recover the ethanol, yielding total iridoid glycosides.

[0007] In step (1), the ratio of honeysuckle berry powder to methanol solution is 1g:20-30mL, and the mass concentration of the methanol solution is 80-85%.

[0008] In step (1), the ultrasonic power is 240-360W.

[0009] In step (2), the macroporous adsorption resin is of type D101, type AB-8, or type XAD-7HP.

[0010] In step (2), the mass concentration of ethanol is 30-50%.

[0011] In step (2), the elution flow rate of ethanol is 5-6 mL / min.

[0012] The total iridoid glycosides extracted by the extraction and purification method described above are used in the preparation of drugs to improve hyperuricemic nephropathy.

[0013] The total iridoid glycosides extracted by the extraction and purification method described above are used in the preparation of health products to improve hyperuricemic nephropathy.

[0014] Application of total iridoid glycosides extracted by the extraction and purification method described above in the preparation of food products that improve hyperuricemic nephropathy.

[0015] The beneficial effects of this invention are: 1. The extraction method of the present invention is simple and easy to operate, suitable for industrial production, and can extract iridoid glycosides from honeysuckle fruit to the maximum extent. Its purification method further increases the content of iridoid glycosides in honeysuckle fruit, which is beneficial for subsequent processing into corresponding pharmaceuticals, health products and food.

[0016] 2. This invention is the first to discover that total iridoid glycosides in honeysuckle fruit can effectively improve hyperuricemic nephropathy, providing a safer approach for the treatment of hyperuricemic nephropathy. Attached Figure Description

[0017] Figure 1 This is a graph showing the content of strychnine at different temperatures in Example 1 of the present invention; Figure 2 This is a graph showing the results of loganin content at different temperatures in Example 1 of the present invention; Figure 3 This is a graph showing the content of strychnine at different ultrasonic powers in Example 1 of the present invention; Figure 4 This is a graph showing the content of loganin at different ultrasonic powers in Example 1 of the present invention; Figure 5 This is a graph showing the content of strychnine in different liquid ratios in Example 1 of the present invention; Figure 6 This is a graph showing the results of loganin content at different material-to-liquid ratios in Example 1 of the present invention; Figure 7 This is a graph showing the content of strychnine at different extraction times in Example 1 of the present invention; Figure 8 This is a graph showing the content of loganin at different extraction times in Example 1 of the present invention; Figure 9 The figures shown in Example 3 of this invention are: Figure A: Statistical graph of mouse body weight change after administration; Figure B: Statistical graph of mouse serum uric acid; ### indicates p < 0.001 compared with the control group; *** indicates p < 0.001 compared with the model group. Figure 10 This is an image showing the HE staining results of kidney tissue from C57BL / 6J mice in Example 3 of this invention; Figure 11 This is a graph showing the results of total iridoid glycosides from honeysuckle fruit improving uric acid-induced HK-2 cell activity in Example 3 of this invention. Detailed Implementation

[0018] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0019] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. Unless otherwise specified, the methods in the following embodiments are conventional methods in the field.

[0020] Example 1: Optimization of the extraction process of honeysuckle fruit (1) Extraction temperature 1.000 g of honeysuckle berry powder was accurately weighed. Extraction temperatures were set at 30℃, 40℃, 50℃, 60℃, and 70℃, with an ultrasonic power of 360 W, a solid-liquid ratio of 1:20 (g:mL), and an extraction time of 30 min. The extract was transferred to a centrifuge tube, centrifuged, and the supernatant was collected and diluted to 25 mL. The solution was filtered through a microporous membrane, and the content was determined by high-performance liquid chromatography (HPLC). The extraction effect was optimal at 50℃. Therefore, a response surface methodology experiment was designed with 50℃ as the center point.

[0021] (2) Ultrasonic power 1.000 g of honeysuckle berry powder was accurately weighed. The extraction temperature was 50 ℃, and the ultrasonic power was set to 180, 240, 300, 360, and 420 W, with a solid-liquid ratio of 1:20 and an extraction time of 30 min. The extract was transferred to a centrifuge tube, centrifuged, and the supernatant was collected and diluted to 25 mL. The solution was filtered through a microporous membrane, and the content was determined by high-performance liquid chromatography (HPLC). The extraction efficiency was highest at an ultrasonic power of 360 W; therefore, a response surface methodology was designed with an ultrasonic power of 360 W as the center point.

[0022] (3) Material-liquid ratio 1.000 g of honeysuckle berry powder was accurately weighed. Extraction was performed at 50 ℃ with an ultrasonic power of 360 W. The solid-liquid ratios were set to 1:10, 15, 20, 25, and 30, with an extraction time of 30 min. The extract was transferred to a centrifuge tube, centrifuged, and the supernatant was collected and diluted to 25 mL. The solution was filtered through a microporous membrane, and the content was determined by high-performance liquid chromatography (HPLC). The extraction efficiency was highest at a solid-liquid ratio of 1:25. Therefore, a response surface methodology was designed with a solid-liquid ratio of 1:25 as the central point.

[0023] Extraction time 1.000 g of honeysuckle berry powder was accurately weighed. Extraction was performed at 50 ℃, with an ultrasonic power of 360 W and a solid-liquid ratio of 1:20. Extraction times were 5, 10, 20, 30, and 40 min. The extract was transferred to centrifuge tubes, centrifuged, and the supernatant was collected and diluted to 25 mL. The solution was filtered through a microporous membrane, and the content was determined by high-performance liquid chromatography (HPLC). The extraction efficiency was highest at an extraction time of 30 min. Therefore, a response surface methodology was designed with an extraction time of 30 min as the center point.

[0024] Based on the results of the single-factor experiment, the following four-factor, three-level Box-Behnken experiment was designed using Design-Expert software, with extraction temperature (A), ultrasonic power (B), material-liquid ratio (C), and extraction time (D) as the factors to be investigated, and strychnine and loganin content (mg / g) as the response values.

[0025] Table 1. Four-factor, three-level experiment

[0026] Based on Design-Expert 13, input the response surface factor level table to obtain the following experimental design table. With extraction temperature (A), ultrasonic power (B), material-liquid ratio (C), and extraction time (D) as independent variables, and strychnine and loganin as response values, the extraction rate Y and regression model variance analysis table are obtained.

[0027] Since strychnine and loganin are the main components of total iridoid glycosides in honeysuckle fruit, strychnine and loganin are used as detection indicators in this method.

[0028] Table 2 Box-Behnken Experimental Design and Results

[0029] Table 3. Response Surface Data Analysis of Strychnosine

[0030] Using Design Expert software, a multiple regression was performed on the table to obtain a binary multivariate regression equation for the strychnine content (Y). The regression model equations for extraction temperature (A), ultrasonic power (B), material-to-liquid ratio (C), and extraction time (D) are as follows: Y=6.402+0.0475A+0.1283B+0.2708C+0.01D+0.02AB-0.025AC+0.0125AD+0.0825BC+0.0025BD+0.065CD-0.231A 2 -0.3872B 2 -0.706C2 -0.1022D 2 .

[0031] As shown in Table 3, the material-to-liquid ratio (C) has the greatest impact on the extraction rate of strychnine, followed by ultrasonic power (B), while the extraction time (A) has the least impact on strychnine.

[0032] Table 4. Response Surface Data Analysis of Strychnosine

[0033] Using Design Expert software, a multiple regression was performed on the table to obtain a binary multivariate regression equation for the strychnine content (Y). The regression model equations for extraction temperature (A), ultrasonic power (B), material-to-liquid ratio (C), and extraction time (D) are as follows: Y=2.126+0.0267A+0.045B+0.0867C+0.01D+0.005AB-0.005AC-0.015AD+0.0175BC+0.0175BD-0.0375CD-0.0817A 2 -0.1842B 2 -0.2617C 2 -0.0668D 2 ; As shown in Table 4, the material-to-liquid ratio (C) has the greatest impact on the extraction rate of strychnine, followed by ultrasonic power (B), while the extraction time (A) has the least impact on strychnine.

[0034] The optimal extraction process for honeysuckle berries, determined using Design Expert, yielded the conditions maximizing strychnine and loganin content: a temperature of 52.045 ℃, ultrasonic power of 371.107 W, a solid-liquid ratio of 1:25.672, and an extraction time of 30.132 min (Table 5). Considering actual production conditions, a temperature of 50 ℃, ultrasonic power of 360 W, a solid-liquid ratio of 1:25, and an extraction time of 30 min were used. Under these conditions, the strychnine content was 6.059 mg / g, and the loganin content was 2.051 mg / g, which are close to the expected values ​​of 6.439 and 2.138 mg / g, respectively.

[0035] Table 5 Optimal Extraction Process for Honeysuckle Fruit

[0036] Example 2: Optimization of the process for purifying total cyclohexene glycosides from honeysuckle fruit using macroporous resin Preparation of the honeysuckle berry adsorption stock solution: Weigh 30 g of honeysuckle berry powder, add 25 times the amount of 80% methanol, heat at 50 ℃ and extract with ultrasonic assistance for 30 min, filter, concentrate the filtrate until no alcohol odor remains, make up to 0.1 g honeysuckle berry powder / mL with pure water, let stand, filter, and obtain the honeysuckle berry adsorption stock solution, which is stored at 4 ℃ for later use. HPLC analysis determined that the concentration of strychnine in the honeysuckle berry adsorption stock solution was 1.65 mg / mL, the content of strychnine was 16.5 mg / g, the concentration of loganin was 0.94 mg / mL, and the content of loganin was 9.4 mg / g.

[0037] Static adsorption and desorption: Take 10 mL of the original adsorption solution of honeysuckle berries, three times. Weigh 1 g each of three different types of macroporous resins (D101, AB-8, and XAD-7HP) into 50 mL Erlenmeyer flasks, add 10 mL of the resin solution to each, shake well, and place in a constant temperature shaker for 12 h (25 ℃, 100 r / min). After filtration, take 20 μL of the supernatant from each flask for HPLC analysis to determine the adsorption rate. Then add 10 mL of 50% ethanol to each flask for desorption, shake well, and place in a shaker for 5 h (25 ℃, 100 r / min). After filtration, take 20 μL of the supernatant from each flask for HPLC analysis to determine the desorption rate.

[0038] Adsorption rate = (concentration of original solution * 10 mL - concentration after adsorption * 10 mL) / (concentration of original solution * 10 mL); Desorption rate = (concentration after desorption * 10 mL) / (concentration of original solution * 10 mL - concentration after adsorption * 10 mL).

[0039] Table 6 Adsorption rates of four different types of macroporous adsorption resins Table 6 Adsorption rates of three different types of macroporous adsorption resins

[0040] Table 7 Desorption rates of three macroporous adsorption resins

[0041] Calculations showed that the adsorption rates of cyclohexene glycosides from honeysuckle on four macroporous adsorption resins were AB-8 type > XAD-7HP type > D101 type, and the desorption rates were AB-8 type > XAD-7HP type > D101 type.

[0042] Three different types of macroporous resins were packed into columns (70 g each), with a column height of 14.5 cm and a retention volume of approximately 90 mL. The columns were washed with water until no alcohol odor remained. Four 50 mL portions of the original honeysuckle berry adsorption solution were loaded onto the columns at a flow rate of 1 mL / min and allowed to stand overnight. Elution was then performed sequentially with 0% (10 BV), 10% (10 BV), 30% (10 BV), 50% (10 BV), 70% (10 BV), and 90% (5 BV) ethanol at a flow rate of 5 mL / min. The eluent was collected, concentrated under reduced pressure, and diluted to 25 mL with water. 5 mL portions of each eluent were placed in a weighing dish, evaporated to dryness, and the mass of each eluted sample was measured. Separately, each eluent was analyzed using high-performance liquid chromatography (HPLC) to determine the contents of loganic acid and loganin. Based on Table 8, AB-8 type macroporous adsorption resin with 30% ethanol as the eluent yielded the highest contents of loganic acid and loganin.

[0043] Table 8. Determination results of strychnine and loganin.

[0044] Example 3: Pharmacological activity study of total iridoid glycosides from honeysuckle fruit The protective effect of total iridoid glycosides from honeysuckle fruit against hyperuricemic nephropathy was studied by in vivo induction of hyperuricemia in C57BL / 6J mice with potassium oxonate and adenine. In vitro cell experiments showed that total iridoid glycosides from honeysuckle fruit had a protective effect against uric acid-induced HK-2 cells.

[0045] (1) Experiment on the reduction of uric acid in mice with hyperuricemia by total iridoid glycosides from honeysuckle fruit. C57BL / 6J mice (male, 18-22 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. After 7 days of acclimatization under alternating light and dark conditions, experiments were conducted. Following 7 days of acclimatization, all mice were randomly divided into two groups: a normal control group (n=10) and a model group (n=20). The model group mice were induced to develop hyperuricemia by gavage with 300 mg / kg / day potassium oxonate (PO) and 50 mg / kg / day adenine (AD). To prepare the solution, potassium oxonate and adenine were finely ground in a mortar and then suspended in a 0.5% carboxymethyl cellulose (CMC-Na) solution. The normal control group mice were gavage with an equal volume of 0.5% CMC-Na solution. All mice were gavaged once daily at 9:00 AM. Two weeks after drug administration, three mice were randomly selected from both the normal group and the model group to measure their serum uric acid levels. If the serum uric acid level in the model group was significantly higher than that in the normal group, the hyperuricemia mouse model was successfully established. After successful model establishment, the model group mice were randomly divided into two groups (n=10 per group): the model group and the total iridoid glycosides group (LCI). As shown in Table 9, each group was administered the corresponding drug by gavage once daily for two weeks. Animal grouping and drug administration are shown in Table 9. Table 9 Animal grouping and administration

[0046] like Figure 9 As shown, the body weight of mice in the model group decreased significantly, and after treatment with 100 mg / kg / d of total iridoid glycosides from *Honeysuckle berries*, the body weight of mice recovered to varying degrees. Compared with the control group (68.57 ± 18.95 μmol / L), the serum uric acid level in the model group increased significantly to 248.75 ± 25.77 μmol / L, indicating that the hyperuricemia mouse model was successfully established. After treatment with total iridoid glycosides from *Honeysuckle berries*, the serum uric acid level of hyperuricemic mice decreased significantly to 82.47 ± 22.28 μmol / L. These results indicate that total iridoid glycosides from *Honeysuckle berries* can effectively reduce serum uric acid levels and improve renal function damage caused by hyperuricemia.

[0047] HE staining method for observing the pathological morphology of kidney tissue: like Figure 10As shown, the control group mice had intact kidney structure, normal glomerular morphology, neatly arranged renal tubular epithelial cells, and no interstitial edema or inflammatory cell infiltration. The model group mice showed obvious pathological damage to their kidneys, with widening of the glomerular capsule space, large-area vacuolar degeneration, necrosis and shedding of renal tubular epithelial cells, obvious urate crystal deposition and protein casts in the lumen, and significant edema and a large number of inflammatory cell infiltration in the renal interstitium. Compared with the model group, the kidney damage of mice in the blueberry iridoid group was significantly improved, the glomerular morphology tended to be normal, the degree of renal tubular epithelial cell degeneration and shedding was greatly reduced, the crystal deposition in the lumen was significantly reduced, the interstitial inflammatory response was significantly alleviated, and the tissue integrity was effectively protected.

[0048] In vitro protection of uric acid-induced HK-2 cells by total iridoid glycosides from honeysuckle fruit. HK-2 cells were cultured in MEM medium supplemented with 10% fetal bovine serum and 1% penicillin + streptomycin, and incubated in a 5% CO2 incubator. A hyperuricemia cell model was established by stimulating HK-2 cells with 600 µmol / L uric acid for 24 h.

[0049] Cell viability assay: HK-2 cells were grown at a rate of 1 × 10⁻⁶ 4 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured for 24 h using standard methods. Cells were then treated with different concentrations of total iridoid glycosides from *Honeysuckle berries* for 12 h. The original culture medium was discarded, and fresh culture medium containing 10% CCK-8 solution was added, followed by 2 h of culture. Immediately after incubation, the absorbance (OD) of each well was measured at 450 nm using a microplate reader, and cell viability was calculated. The optimal concentrations of total iridoid glycosides from *Honeysuckle berries* were determined to be 12.5, 25, and 50 μM.

[0050] HK-2 cells were processed at a rate of 1 × 10 4 HK-2 cells were seeded at a density of 1 cell / well in 96-well plates and cultured for 24 h using standard methods. A hyperuricemia cell model was established by stimulating HK-2 cells with 100 mg / L uric acid for 24 h. Cells were then treated with different concentrations of total iridoid glycosides from *Honeysuckle berries* for 12 h, followed by 2 h of culture in fresh medium containing 10% CCK-8 solution. Immediately after incubation, the absorbance (OD) of each well was measured at 450 nm using a microplate reader, and cell viability was calculated. Figure 11 The results showed that, compared with the normal control group, the cell viability in the model group was significantly reduced, and the difference was statistically significant. P <0.05); Compared with the model group, both medium and high doses in the treatment group significantly increased the cell viability of HK-2 cells ( P<0.05), total iridoid glycosides from honeysuckle fruit have a significant protective effect against uric acid-induced HK-2 cells.

[0051] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

Claims

1. A method for extracting and purifying total iridoid glycosides from honeysuckle fruit, characterized in that, Includes the following steps: (1) Extraction: Weigh out honeysuckle berry powder, add methanol solution, heat to 40-60℃, extract with ultrasonic assistance for 20-40 min, filter, concentrate under reduced pressure to recover methanol, and obtain honeysuckle berry extract; (2) Purification: The extract of honeysuckle fruit was subjected to macroporous adsorption resin column chromatography, eluted with ethanol, and concentrated under reduced pressure to recover the ethanol, yielding total iridoid glycosides.

2. The extraction and purification method according to claim 1, characterized in that, In step (1), the ratio of honeysuckle berry powder to methanol solution is 1g:20-30mL, and the mass concentration of the methanol solution is 80-85%.

3. The extraction and purification method according to claim 1, characterized in that, In step (1), the ultrasonic power is 240-360W.

4. The extraction and purification method according to claim 1, characterized in that, In step (2), the macroporous adsorption resin is of type D101, type AB-8, or type XAD-7HP.

5. The extraction and purification method according to claim 1, characterized in that, In step (2), the mass concentration of ethanol is 30-50%.

6. The extraction and purification method according to claim 1, characterized in that, In step (2), the elution flow rate of ethanol is 5-6 mL / min.

7. The use of the total iridoid glycosides extracted by the extraction and purification method described in claim 1 in the preparation of drugs for improving hyperuricemic nephropathy.

8. The application of the total iridoid glycosides extracted by the extraction and purification method described in claim 1 in the preparation of health products for improving hyperuricemic nephropathy.

9. The application of the total iridoid glycosides extracted by the extraction and purification method described in claim 1 in the preparation of food products that improve hyperuricemic nephropathy.

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