Eutectic solvent extraction method and application of pigeon pea leaf polysaccharide

Abstract

The invention discloses a deep-eutectic solvent extraction method of pigeon pea leaf polysaccharide and application of the pigeon pea leaf polysaccharide. Belongs to the technical field of natural active ingredients. The deep eutectic solvent is prepared by taking choline chloride as a hydrogen bond receptor and different substances as hydrogen bond donors, the extraction process is optimized by combining a single factor experiment with a response surface method, and the activity is evaluated by adopting a zebra fish osteoporosis model and an MC3T3-E1 cell model. Results show that the optimal DES is choline-1, 3-butanediol (the molar ratio is 1: 5, and the water content is 40%), the optimal extraction process is that the material-liquid ratio is 1: 49, the extraction temperature is 89 DEG C, the extraction time is 78min, and at the moment, the CCP yield reaches 8.47%. An activity experiment shows that the CCP can increase the mineralization area of the skull and the spine of the osteoporotic zebra fish and improve the alkaline phosphatase activity of MC3T3-E1 cells and the number of mineralized nodules. The invention provides reference for efficient extraction of CCP and research and development of anti-osteoporosis drugs.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of natural active ingredients, and more particularly to a deep eutectic solvent extraction method of Caesalpinia japonica leaf polysaccharide and application thereof. BACKGROUND

[0002] Caesalpinia japonica leaf is the dry leaf of Caesalpinia japonica of Leguminosae and is a traditional medicinal material in Guangdong, Nan and other places. Cajanus Cajan Caesalpinia japonica leaf has the biological activities of lowering blood sugar, lowering blood lipid and protecting cardiovascular and cerebrovascular damage. Caesalpinia japonica leaf contains flavonoids, stilbenes, polysaccharides, steroids and other chemical components, and the polysaccharides are considered to be one of the important effective components of Caesalpinia japonica leaf. Compared with the traditional water extraction method, the deep eutectic solvent (DES) extraction method has the advantages of high extraction efficiency and structural integrity. However, there are few reports on the deep eutectic solvent extraction method of Caesalpinia japonica leaf polysaccharide, and there are few studies on the new application thereof.

[0003] Therefore, how to provide a deep eutectic solvent extraction method of Caesalpinia japonica leaf polysaccharide and study the new application thereof is a technical problem to be solved by those skilled in the art. SUMMARY

[0004] Therefore, the application provides a deep eutectic solvent extraction method of Caesalpinia japonica leaf polysaccharide and application thereof.

[0005] The application optimizes the process of extracting Caesalpinia japonica leaf polysaccharide (Polysaccharide, CCP) by using a deep eutectic solvent, thereby providing a reference for the enrichment of the medicinal components of Caesalpinia japonica leaf and the improvement of the production process of related drugs. Cajanus Cajan

[0006] To solve the above technical problems, the application adopts the following technical solutions. A deep eutectic solvent extraction method of Caesalpinia japonica leaf polysaccharide, wherein the deep eutectic solvent is a deep eutectic solvent formed by taking choline chloride as a hydrogen bond acceptor and 1,3-butanediol as a hydrogen bond donor.

[0007] Further, the molar ratio of choline chloride to 1,3-butanediol is 1:5.

[0008] Further, the solid-liquid ratio is 1: (40-60), the extraction temperature is 80-100 DEG C, and the extraction time is 60-100 min.

[0009] Further, the solid-liquid ratio is 1:49, the extraction temperature is 89 DEG C, and the extraction time is 78 min.

[0010] Further, the water content of the deep eutectic solvent is 40%.

[0011] ​The Caesalpinia minifolia leaf polysaccharide prepared by the extraction method.

[0012] The Caesalpinia minifolia leaf polysaccharide is applied to preparation of an anti-osteoporosis drug.

[0013] Further, the CCP can increase the mineralization area, improve the alkaline phosphatase activity, and increase the number of mineralized nodules.

[0014] Compared with the prior art, the technical solution has the following beneficial effects: The low eutectic solvent is prepared by taking choline chloride as a hydrogen bond acceptor and different substances as hydrogen bond donors, the extraction process is optimized by single factor experiment combined with response surface method, and the activity is evaluated by using the osteoporosis model of zebrafish and the MC3T3-E1 cell model. The results show that the optimal DES is choline chloride-1,3-butanediol (molar ratio 1:5, water content 40%), the optimal extraction process is that the solid-liquid ratio is 1:49, the extraction temperature is 89 DEG C, and the extraction time is 78 min, and the CCP yield reaches 8.47% at this time. The activity experiment shows that the CCP can increase the mineralization area of the head bone and spine of the osteoporosis zebrafish, improve the alkaline phosphatase activity of the MC3T3-E1 cell, and increase the number of mineralized nodules. The application provides a reference for efficient extraction of CCP and research and development of anti-osteoporosis drugs. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only embodiments of the present application, and those skilled in the art can obtain other drawings according to the provided drawings without creating any inventive labor.

[0016] Figure 1 The influence of different DESs on the polysaccharide yield in the embodiment 1 of the present application; Figure 2 The influence of different molar ratios on the polysaccharide yield in the embodiment 1 of the present application; Figure 3 The influence of different water contents on the polysaccharide yield in the embodiment 1 of the present application; Figure 4 The influence of different solid-liquid ratios on the polysaccharide yield in the embodiment 1 of the present application; Figure 5 The influence of different extraction temperatures on the polysaccharide yield in the embodiment 1 of the present application; Figure 6 The influence of different extraction times on the polysaccharide yield in the embodiment 1 of the present application; Figure 7 The influence of different concentrations of CCP on the osteoporosis zebrafish in the embodiment 1 of the present application; Figure 8 Effects of different concentrations of CCP on the viability of MC3T3-E1 Subclone14 cells in Example 1 of the present application; Figure 9 Effects of different concentrations of CCP on the ALP staining of MC3T3-E1 Subclone14 cells in Example 1 of the present application; Figure 10 Effects of different concentrations of CCP on the alizarin red staining of MC3T3-E1 Subclone14 cells in Example 1 of the present application. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0018] The experimental materials required by the present application are conventional experimental materials, which are purchased from commercial channels. The experimental methods not mentioned are conventional experimental methods, which will not be described one by one here.

[0019] Example 1 1 Materials 1.1 Experimental animals and cells AB type zebrafish parents were purchased from Shandong Yixiyue Biological Technology Co., Ltd. and were bred and reproduced by the laboratory; MC3T3-E1 Subclone14 (item number: FH0382) was provided by Shanghai Fuheng Biological Co., Ltd.

[0020] 1.2 Drugs and reagents Cajanus cajan leaves were provided by Guangxi Wushengyuan Agricultural Development Co., Ltd. (batch number 20231115); Choline chloride (purity ≥98%), ethylene glycol (purity ≥99%), urea (purity ≥99%), lactic acid (purity ≥98%), 1,3-butanediol (purity ≥99%), 1,4-butanediol (purity ≥98%), glycerol (≥99%), and anhydrous ethanol were purchased from Yunnan KeYiHua Glass Co., Ltd. Alendronate sodium, alizarin red, and phenol were purchased from Shanghai Macklin Biochemical Technology Co., Ltd. Concentrated sulfuric acid, n-butanol, and chloroform were purchased from Chengdu Kolon Chemicals Co., Ltd. D(+) anhydrous glucose was purchased from Beijing Zhongke Huabiao Biological Technology Research Institute; Dialysis bags (3500) were purchased from Shanghai Yuanye Biological Technology Co., Ltd. L-ascorbic acid, sodium β-glycerophosphate, dexamethasone, prednisolone were purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. 3-Aminobenzoic acid ethyl ester methanesulfonate (Adamas), NaCl, KCl, CaCl2·6H2O, MgSO4·7H2O, KOH were purchased from Tianjin Damao Chemical Reagent Factory; H2O2 was purchased from Kunming Tailikang Industry and Trade Co., Ltd. MEM-α medium (gibco), fetal bovine serum were purchased from Shanghai Dart Hill Biological Technology Co., Ltd. 4% paraformaldehyde, penicillin-streptomycin solution were purchased from Anhui Baishaji Biological Technology Co., Ltd. CCK8 kit was purchased from Suzhou Youyilanbi Biological Technology Co., Ltd. PBST, DMSO, BCIP / NBT alkaline phosphatase color reagent kit were purchased from Solabio; Osteoblast mineralization nodule staining kit was purchased from Shanghai Biyun Tian Biological Technology Co., Ltd.

[0021] 1.3 Instruments and equipment WGLL-125BE electric heating air drying oven (Tianjin Test Instrument Co., Ltd.); HZ-124 / 85S electronic balance (Huazhi (Fujian) Electronic Technology); HWS-26 constant temperature water bath (Shanghai Yiheng Scientific Instruments Co., Ltd.); DF-101S heat collecting magnetic stirrer (Shanghai Labline Instrument Co., Ltd.); BS223S high-speed refrigerated centrifuge (Beijing Sartorius Instrument System Co., Ltd.); ER-52A rotary evaporator (Shanghai Yalong Biochemical Instrument Factory); UH5300 ultraviolet visible spectrophotometer (Hitachi High-Technologies Corporation); B15-10NDA0002 freeze dryer (Ningbo Xinzhi Biological Technology Co., Ltd.); Zebrafish breeding system (Beijing Aiseng Technology Development Co., Ltd.); GDN-300D-4 light incubator (Ningbo Southeast Instrument Co., Ltd.); ZOOM-2890 body fluorescence microscope (Shanghai Wumao Optical Instrument Co., Ltd.); IX73 research grade inverted microscope (OLYMPUS); Rikang CO2 electric heating water-isolating type cell constant temperature incubator (Shanghai Zhenjie Experimental Equipment Co., Ltd.).

[0022] 2 Experimental method 2.1 Preparation of eutectic solvent Choline chloride as Hydrogen Bond Acceptor (HBA), ethylene glycol, urea, lactic acid, 1,3-butanediol, 1,4-butanediol, glycerol as Hydrogen Bond Donor (HBD) were mixed according to Table 1, a fixed proportion of water was added, heated to 70°C in a magnetic heating stirrer until clear and transparent, then transferred to a glass container and sealed for use. The prepared DES was respectively recorded as DES1~DES6.

[0023] Table 1 Composition of DES

[0024] 2.2 Preparation of Caesalpinia cuatritata leaf polysaccharide (CCP) Caesalpinia cuatritata leaf was dried at 40°C, crushed and sieved through a 40-mesh sieve and then sealed and stored. 1.2 g of Caesalpinia cuatritata leaf powder was weighed into a 100 mL triangular flask, and DES solution was added at a ratio of 40:1 (V / m) and mixed evenly. It was heated in a water bath at 80°C for 40 min, then transferred to a 50 mL centrifuge tube, centrifuged at 4000 rpm for 10 min at 4°C, the supernatant was taken, 4 times the volume of anhydrous ethanol was added, and it was left overnight at 4°C, then centrifuged at 4000 rpm for 10 min at 4°C. The obtained precipitate was dissolved with hot water and then eluted with a macroporous resin. The eluate was concentrated to a certain volume, n-butanol: chloroform (1:4) was added to remove protein until no precipitate was precipitated. Then it was placed in a dialysis bag (molecular weight cut-off 3.5 KDa) for dialysis for 48 h, and then freeze-dried to obtain CCP.

[0025] 2.3 Single factor experiment of Caesalpinia cuatritata leaf polysaccharide extraction Based on the polysaccharide yield, the DES with the highest CCP yield was selected, and single factor experiments were conducted on the basis of water content 50%, solid-liquid ratio 1:40, extraction temperature 80°C, and extraction time 40 min. The effects of DES molar ratio (1:1, 1:2, 1:3, 1:4, 1:5, 1:6), water content (0, 10%, 20%, 30%, 40%, 50%), solid-liquid ratio (1:20, 1:30, 1:40, 1:50, 1:60, 1:70), extraction temperature (50, 60, 70, 80, 90, 100°C), and extraction time (20, 40, 60, 80, 100, 120 min) on the polysaccharide yield were investigated.

[0026] 2.4 Response surface experiment design Based on the results of the single factor experiment, the solid-liquid ratio (A), extraction temperature (B), and extraction time (C) were selected as independent variables, and the polysaccharide yield was used as the index for investigation. The factor levels are shown in Table 2.

[0027] Table 2 Response Surface Analysis Factors and Level Design

[0028] 2.5 Preparation of standard curve and determination of polysaccharide content in pigeon pea leaves The polysaccharide content was determined using the phenol-sulfuric acid method. Accurately measure 0.4, 0.8, 1.2, 1.6, and 2.0 mL of the reference solution into 10 mL stoppered test tubes. Add water to a final volume of 2 mL, then add 1 mL of 0.6% phenol solution, shake well, and quickly add 5 mL of concentrated sulfuric acid. Shake well again, place the tubes in a boiling water bath, and cool to room temperature. Using the corresponding reagents as blanks, measure the absorbance at a wavelength of 490 nm using spectrophotometry. Plot glucose concentration on the x-axis and absorbance on the y-axis to obtain the standard curve equation: y = 0.016x + 0.0137 (R² = 0.9991).

[0029] Dissolve an appropriate amount of CCP in pure water, measure the absorbance value using the method described above, and then substitute it into the standard curve to calculate the mass concentration of pigeon pea leaf polysaccharide.

[0030] CCP yield is calculated using the formula: Polysaccharide yield (%) = (Polysaccharide mass concentration × Sample liquid volume × Dilution factor) / Pigeon pea leaf mass × 100.

[0031] 2.6 Investigation into Anti-Osteoporosis Activity 2.6.1 Zebrafish rearing and embryo collection Six-month-old wild-type AB zebrafish were cultured in an environment of (28.0±1)℃, with 14 hours of light and 10 hours of darkness, pH=6.8~7.5, and sufficient oxygen, and fed twice a day. Suitable-aged breeding zebrafish were selected and allowed to freely chase, mate, and spawn. Healthy eggs laid within 30 minutes were collected. The selected eggs were cleaned and evenly placed in sterile petri dishes with a diameter of 10cm. 20mL of E3 medium was added, and the dishes were incubated in a constant temperature incubator at (28.5±0.2)℃.

[0032] 2.6.2 Screening of Pigeon Bean Leaf Polysaccharide Dosage Concentration Fish eggs with normal development and fertilization for 5 hours were randomly selected and placed into 24-well plates, 20 eggs per well. Different concentrations of CCP solution (50, 100, 150, 200, 250 μg / mL) were prepared with E3 water, and 2 mL of the culture medium of each concentration was added to each well. Each group had 3 replicates. The medium was changed every 24 hours, and the culture period was 9 days. Embryo development and survival were observed.

[0033] 2.6.3 Establishment and grouping of a zebrafish osteoporosis model The experiment was divided into blank group, prednisolone group (25 μmol / L), alendronate sodium group (30 μmol / L), CCP group (50, 100, 200, 400, 800 μg / mL). The 72h developed zebrafish larvae were randomly distributed in 12-hole plates, 20 tails per hole, 2 holes as a group. Add 3mL of the corresponding culture solution to each hole, change the liquid every 24h, and the culture period is 9 days. The skull staining area and the number of spine of the prednisolone group are significantly reduced compared with the blank group, indicating that the modeling is successful.

[0034] 2.6.4 Zebrafish bone staining and analysis Staining of the modeled and administered zebrafish: ①Anesthetize to death with anesthetics.

[0035] ②Discard the anesthetics, fix with 4% paraformaldehyde for 30min (4°C).

[0036] ③Discard the fixative, dehydrate with 50% ethanol for 10-30min.

[0037] ④Remove the dehydrating agent and wash with PBST 3 times (each washing time should not be too long).

[0038] ⑤Add 1% KOH and 1% hydrogen peroxide mixture to bleach for 30min, try not to exceed 45min, and the process needs to be operated in the dark. Observe the staining under a microscope every 15min.

[0039] ⑥Discard the bleaching solution, wash with PBST 3 times, and add 0.005% alizarin red for staining for more than 8h.

[0040] ⑦After staining, remove the staining solution and wash away the excess staining solution with PBST. Place the zebrafish larvae in 0.5% KOH:glycerol (3:1) for 6-8h, 0.5% KOH:glycerol (1:1) for 6-8h, and 0.5% KOH:glycerol (1:3) for 6-8h. After permeation, store the zebrafish larvae in glycerol.

[0041] ⑧Fix the zebrafish larvae in glycerol under a body microscope, collect the head and spine staining area, and count the number of vertebral bone segments.

[0042] 2.6.5 Cell culture Prepare according to MEM-α medium:fetal bovine serum:streptomycin (90:10:1) for the culture of MC3T3-E1 Subclone14 cell line. Culture the cells in a cell culture incubator at 37°C and 5% CO2. When the cells grow and fuse to 80%-90%, use trypsin digestion and subculture for subsequent experiments. The cells used for staining are 5×10 4Each well was inoculated in 12-well plates, and after 24 h of culture, the osteogenic induction medium containing drugs (10 -8 mol / L dexamethasone, 10 mmol / L β-glycerophosphate sodium, and 50 μmol / L ascorbic acid) was replaced every 3 days. The groups were as follows: blank group (osteogenic induction medium), dexamethasone group (10 μmol / L), alendronate sodium group (10 μmol / L), and CCP group (25, 50, and 100 μg / mL).

[0043] 2.6.6 Cell viability detection Each well was inoculated in 96-well plates with 3000 MC3T3-E1 Subclone14 cells, and after 24 h of culture in an incubator, different concentrations of CCP (12.5, 25, 50, 100, 200, 400, and 800 μg / mL) were added for intervention, with 3 replicate wells in each group. Cell viability was determined using a CCK8 kit at 24 h and 48 h, respectively. After adding 10 μL of CCK8 reagent to each well, incubation was performed at 37°C for 1 h, and then the absorbance value of each well was determined on a microplate reader (wavelength 450 nm). Cell viability was calculated according to the CCK8 kit instructions.

[0044] 2.6.7 Alkaline phosphatase (ALP) staining After 7 days of culture with different groups of drugs, the culture medium was aspirated, washed with PBS, and fixed with 4% paraformaldehyde at room temperature for 30 min. PBS was washed 2-3 times, and the required volume of staining solution was prepared according to the BCIP / NBT alkaline phosphatase color reagent kit instructions. After adding an appropriate volume of staining solution, staining was performed at room temperature in the dark until color development. The staining solution was removed, and PBS was washed until the PBS was colorless. Observation and photographing were performed using an inverted microscope.

[0045] 2.6.8 Alizarin red staining After 21 days of culture with different groups of drugs, the culture medium was aspirated, washed with PBS, and fixed with 4% paraformaldehyde at room temperature for 30 min. PBS was washed 2-3 times, and the osteoblast mineralization nodule staining kit was used for staining at room temperature in the dark for 20 min. The staining solution was removed, and pure water was used for washing until the pure water was colorless. Observation and photographing were performed using an inverted microscope.

[0046] 2.7 Data processing All experiments were repeated 3 times, and the results were averaged. Data were processed and analyzed using GraphPad Prism 10.1.2, Design-Expert 13, Origin 2024, Excel, and other software.

[0047] 3 Results 3.1 DES Filtering Depend on Figure 1 It is evident that the composition of DES significantly impacts CCP yield. DES2 (choline chloride-1,3-butanediol) exhibits a higher CCP yield than the other five. This may be due to the moderate strength of the hydrogen bond network formed by the two 1,3-hydroxyl groups of 1,3-butanediol in DES2, which precisely disrupts plant cell walls, allowing polysaccharides to flow out. Furthermore, the 4-carbon chain structure of 1,3-butanediol is compatible with the repeating units of the polysaccharide, and its polarity matches the polysaccharide, ensuring high solubility. Additionally, the DES formed by 1,3-butanediol and choline chloride has low viscosity and better flowability, effectively promoting solvent penetration, increasing the contact area between the polysaccharide and solvent, and improving extraction efficiency. Therefore, DES2 was chosen for subsequent extraction process optimization.

[0048] 3.2 Single-factor experiment 3.2.1 Molar ratio Depend on Figure 2 It is observed that the CCP yield reaches its maximum when the DES molar ratio is 1:5. As the DES molar ratio increases, the CCP yield increases; a higher proportion of 1,3-butanediol results in lower DES viscosity, which facilitates solvent diffusion within the pigeon pea leaf cell wall and dissolves highly polar polysaccharides. When the molar ratio exceeds 1:5, the CCP yield begins to decrease, indicating a decrease in DES polarity and reduced solubility for polysaccharides. Therefore, a choline chloride:1,3-butanediol molar ratio of 1:5 was chosen to investigate other factors.

[0049] 3.2.2 Moisture content Depend on Figure 3 It is observed that when the water content of DES is between 0% and 20%, the CCP yield gradually increases with increasing water content. When the water content reaches 30%, the CCP yield decreases, reaching its maximum at 40%. With further increases in water content, the CCP yield decreases again. Water, as a polar small molecule, has a bidirectional effect on the DES system. With increasing water content, the viscosity of DES decreases, increasing mass transfer efficiency and thus improving the yield. At a water content of 20% to 30%, the number of water molecules reaches a "critical interference value," forming hydrated chloride ions with choline chloride, disrupting the original hydrogen bond network and causing a decrease in yield. At a water content of 40%, sufficient water molecules form "bridging hydrogen bonds," and the viscosity of DES further decreases, achieving an optimal balance between dissolution and mass transfer efficiency. When the water content exceeds 40%, a large number of water molecules form a dense hydrated layer on the CCP, causing the DES properties to be lost. Therefore, a water content of 40% is chosen for further investigation of other factors.

[0050] 3.2.3 Material-to-liquid ratio Depend on Figure 4It can be seen that the yield of CCP in the extract first increases and then decreases with the increase of the solid-liquid ratio. The highest CCP yield is achieved when the solid-liquid ratio is 1:60. This is because when the solid-liquid ratio is low, the solvent and pigeon pea leaves cannot fully contact each other, resulting in less dissolved product. As the solid-liquid ratio increases, the contact area between CCP and the solvent increases, increasing the dissolution of polysaccharides. However, when the amount of solvent is too large, the intermolecular forces between DES and polysaccharides weaken, causing the CCP yield to begin to decrease. Since the CCP yield fluctuates relatively little between 1:50, 1:60, and 1:70, considering solvent conservation, a solid-liquid ratio of 1:50 was chosen for further investigation of subsequent factors.

[0051] 3.2.4 Extraction Temperature like Figure 5 It is observed that within the extraction temperature range of 50℃ to 90℃, the CCP yield gradually increases with increasing temperature, reaching its highest at 90℃. This is likely because as temperature increases, the viscosity of DES decreases, increasing its fluidity and accelerating polysaccharide dissolution. However, when the extraction temperature exceeds 90℃, the high temperature damages the polysaccharide structure and the stability of DES, thus reducing the extraction rate. Therefore, the optimal extraction temperature is 90℃.

[0052] 3.2.5 Extraction Time Depend on Figure 6 It can be seen that the CCP yield first increases and then decreases with the extension of extraction time. Between 20 and 80 min, the yield increases with increasing extraction time, possibly because the polysaccharides cannot be completely released in a short time. However, with prolonged extraction time, more polysaccharides are dissolved and released into the DES, thus continuously increasing the polysaccharide yield. After 80 min, the yield begins to decrease, possibly due to the degradation of the CCP structure caused by excessive extraction time. This indicates that excessively long extraction time is not conducive to CCP extraction; therefore, an extraction time of 80 min was chosen for further investigation of subsequent factors.

[0053] 3.3 Response Surface Experiment 3.3.1 Response surface methodology results and analysis of variance Based on the results of the single-factor experiments, a three-factor, three-level response surface optimization experiment was constructed using the material-to-liquid ratio (A), extraction temperature (B), and extraction time (C) as three factors, and the yield of pigeon pea leaf polysaccharides (Y) as the indicator. The Box-Behnke design (BBD) method in Design-Expert 13 software was employed. The results are shown in Table 3. This experimental design consisted of 17 groups, including 12 factorial experiments and 5 repeated experiments. Regression fitting analysis was performed on the experimental results in Table 3, yielding the quadratic regression equation: Y = 8.83 - 0.0461A - 0.1582B - 0.1046C + 0.1255AB - 0.2463AC - 0.0740BC - 0.3972A 2-1.98B 2 -0.4672C 2 .

[0054] Table 3 Response surface analysis experimental design and results

[0055] Table 4 Variance analysis results of response surface fitting regression equation

[0056] As shown in Table 4, the model P<0.0001, with extremely significant, simulation equation determination coefficient R 2 =0.9887, R 2 adj=0.9742, the coefficient of variation (CV) is 2.41% (<10%), indicating good accuracy, the P value of loss fitting term is 0.8327, not significant, indicating that the fitting effect and prediction ability of the quadratic polynomial regression model is good, and the experimental error is small. The P value of B-extraction temperature in the first order term (0.0212) is less than 0.05, indicating that the extraction temperature has a significant effect on the yield of CCP; the interaction term AC (P=0.0140) is significant, indicating that the interaction of the solid-liquid ratio and the extraction time has a significant effect on the yield of CCP; the quadratic term A 2 (P=0.0010), B 2 (P<0.0001), C 2 (P=0.0004) is extremely significant, indicating that the influence of each factor on the yield of CCP shows a complex nonlinear relationship. As shown by the F value, the influence degree of each factor on the yield of CCP is in the order of B-extraction temperature>C-extraction time>A-solid-liquid ratio.

[0057] 3.3.2 Verification of the optimal extraction process Through the analysis of Design-Expert13 software, the optimal extraction process parameters of CCP were determined as follows: solid-liquid ratio 1:49.66, extraction temperature 89.60℃, and extraction time 78.01 min. Under these conditions, the predicted value of the yield of CCP was 8.83%. In order to facilitate experimental operation, the extraction process parameters were adjusted to solid-liquid ratio 1:49, extraction temperature 89℃, and extraction time 78 min. The verification experiment results showed that the average yield of CCP measured by three parallel experiments was 8.47%, which was close to the predicted value, and the relative standard deviation of the two was 2.07%, indicating that the extraction process optimized by the response surface method was stable and reliable.

[0058] 3.4 Growth and development of zebrafish larvae After the zebrafish were cultured with culture solution containing different concentrations of CCP, they were observed under a microscope. The embryos had normal morphology and developed into juvenile fish normally at 72h and 96h. Compared with the blank group, the juvenile fish in each CCP group did not show death or obvious deformity. Therefore, it is believed that CCP at a concentration of 800 μg / mL or below has no effect on the growth of juvenile fish. Subsequent experiments were conducted to explore the efficacy of CCP at concentrations of 100, 150 and 200 μg / mL.

[0059] 3.5 Zebrafish Alizarin Red Staining Microscopic imaging of the skull and spine of 9dpf zebrafish in each group after alizarin red staining is shown in Figure 7 The mineralized area of the skull and spine of zebrafish is shown in the figure. Compared with the blank group, the mineralized area of the skull and spine of zebrafish in the prednisolone group was significantly reduced, indicating that the prednisolone-induced zebrafish osteoporosis model was successful. Compared with the prednisolone group, the mineralized area of the skull and spine of zebrafish in the CCP group was significantly increased in a dose-dependent manner.

[0060] 3.6 Effect of CCP on cell viability After the cells were administered, cell viability was detected at 24h and 48h, and the results are shown in Figure 8 In the 24h and 48h cell viability determination experiments, CCP at a concentration of 200 μg / mL had a significant effect on cell viability. Therefore, CCP at a concentration of 100 μg / mL or below was selected for subsequent experiments.

[0061] 3.7 ALP Staining of MC3T3-E1 Subclone14 Cells The results of ALP staining are shown in Figure 9 After CCP intervention for 7 days, the ALP staining positive rate of cells in the dexamethasone group was significantly lower than that in the blank group, indicating that the modeling was successful. The ALP staining positive rate of cells in each CCP group was significantly higher than that in the dexamethasone group in a dose-dependent manner, indicating that CCP had an improving effect on OP.

[0062] 3.8 Alizarin Red Staining of MC3T3-E1 Subclone14 Cells The results of alizarin red staining are shown in Figure 10 After CCP intervention for 21 days, the mineralized nodule staining in the dexamethasone group was significantly less than that in the blank group, indicating that the modeling was successful. The mineralized nodule staining in each CCP group was better than that in the dexamethasone group in a dose-dependent manner, indicating that CCP could effectively promote the mineralization of MC3T3-E1 Subclone14 cells, thereby treating OP.

[0063] 4 Discussion and Conclusion The application constructs a DES system to extract CCP, and optimizes the extraction process through single factor test and response surface analysis method. The results show that: the optimal solvent system is choline chloride: 1,3-butanediol (molar ratio 1:5), the optimal extraction conditions are 40% water content, 1:49 solid-liquid ratio, 89 DEG C extraction temperature and 78 min extraction time. Under the optimal conditions, the actual extraction rate of CCP is 8.47%, which is close to the predicted value 8.83%, which verifies the stability and feasibility of the model. The pharmacological effect is explored through zebrafish and MC3T3-E1 cell osteoporosis model. The zebrafish is subjected to alizarin red staining, and the MC3T3-E1 Subclone14 cells are subjected to alkaline phosphatase staining and alizarin red staining. The results show that the CCP extracted by DES can promote the secretion of alkaline phosphatase and increase the deposition of calcium in the bone, that is, the CCP extracted by DES can promote the differentiation and mineralization of osteoblasts, which suggests that CCP can improve OP through this way. The application not only establishes an efficient and environmentally friendly CCP extraction method, but also reveals the potential anti-OP function of CCP.

[0064] The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the difference from other embodiments, and the same or similar parts between the various embodiments can be referred to each other.

[0065] The above description of the disclosed embodiments enables a person skilled in the art to implement or use the application. Various modifications to the embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the application. Therefore, the application will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for eutectic solvent extraction of polysaccharides from pigeon pea leaves, characterized in that, The eutectic solvent uses choline chloride as the hydrogen bond acceptor and 1,3-butanediol as the hydrogen bond donor.

2. The extraction method as described in claim 1, characterized in that, The molar ratio of choline chloride to 1,3-butanediol is 1:

5.

3. The extraction method as described in claim 1, characterized in that, The material-to-liquid ratio is 1:(40~60), the extraction temperature is 80~100℃, and the extraction time is 60~100min.

4. The extraction method as described in claim 1, characterized in that, The material-to-liquid ratio was 1:49, the extraction temperature was 89℃, and the extraction time was 78 min.

5. The extraction method as described in claim 1, characterized in that, The water content of the eutectic solvent is 40%.

6. The pigeon pea leaf polysaccharide prepared by the extraction method according to any one of claims 1 to 5.

7. The use of the pigeon pea leaf polysaccharide according to claim 6 in the preparation of an anti-osteoporosis drug.

8. The application as described in claim 7, characterized in that, It is used to increase the mineralized area, enhance alkaline phosphatase activity, and increase the number of mineralized nodules.

Images