Icariside II compound preparation for protecting PC12 cell injury and preparation method thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to an icariside II compound preparation for protecting PC12 cell injury and a preparation method thereof. Preparation raw materials of the compound preparation comprise the following components: modified icariside II and purslane source vesicles. The invention further provides a preparation method of the compound preparation. The prepared compound preparation can effectively protect PC12 cell damage.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to an icariin II complex preparation for protecting PC12 cells from damage and its preparation method. Background Technology

[0002] One of the core pathological mechanisms of neurodegenerative diseases (such as cerebral ischemia-reperfusion injury, Alzheimer's disease, and Parkinson's disease) is neuronal damage and apoptosis caused by factors such as ischemia, hypoxia, and oxidative stress. The PC12 cell line (rat adrenal pheochromocytoma cells), due to its neuron-like characteristics, can differentiate under specific conditions and express various neurotransmitters and receptors, and is widely used in in vitro simulations of neuronal injury and in studying the mechanisms of neuroprotective drugs. Epimedium, a plant belonging to the genus Epimedium of the Berberidaceae family, has the effects of strengthening muscles and bones, warming and tonifying the body, and dispelling wind and dampness. It is used to treat rheumatic pain, hemiplegia, and impotence. Icariin II (ICS II) is one of the main active components of Epimedium, but its pharmacological effects have been relatively limited. Studies have shown that ICS II can alleviate cerebral microcirculatory disturbances and neuronal damage in the CA1 region of the hippocampus in a gerbil model of cerebral ischemia-reperfusion injury. Its mechanism of action may be related to enhancing the activity of mitochondrial respiratory chain complex I and reducing ROS generation. There is currently limited research on whether ICS II can combat PC12 cell damage. This invention aims to provide pharmacological evidence for the use of ICS II in neuroprotection. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention proposes a picaridin II complex preparation for protecting PC12 cells from damage and its preparation method.

[0004] This invention is achieved through the following technical solution: An icariin II complex preparation for protecting PC12 cells from damage, the raw materials for preparation include the following components: modified icariin II and purslane-derived vesicles.

[0005] Furthermore, the method for preparing the purslane-derived vesicles includes the following steps: L1. After cleaning the purslane, cut it into small pieces and put it into a juicer. Add pre-cooled PBS solution to cover the purslane. Juice at 4°C for 1 min, stop for 1 min, and repeat 5 times. Filter the purslane juice with gauze to remove the residue. Centrifuge the filtrate and collect the supernatant. L2. Take the supernatant obtained in step L1, filter it through a 0.22 μm filter membrane, centrifuge it at high speed, resuspend the precipitate in PBS solution, and perform sucrose gradient purification on the resuspended solution. Collect 45-60% of the banded portion, wash it three times with PBS solution, resuspend it in PBS solution, freeze dry it, and obtain purslane-derived vesicles.

[0006] Further, in step L1, the centrifugation is performed at 4°C, centrifuging at 1000×g for 10 min, taking the supernatant and centrifuging at 3000×g for 20 min, and then taking the supernatant again and centrifuging at 10000×g for 30 min.

[0007] Furthermore, in step L2, the high-speed centrifugation is performed at 4°C for 1.5-2 hours at 100,000 × g.

[0008] Further, in step L2, the sucrose gradient purification is performed by transferring the resuspension to a sucrose gradient solution (8%, 30%, 45%, and 60%, dissolved in 20 mM Tris-Cl at pH 7.2) and centrifuging at 4°C and 150,000 × g for 2 h.

[0009] Furthermore, the present invention also provides a method for preparing the icariin II complex preparation for protecting PC12 cell damage, comprising the following steps: S1. Icariin II was dissolved in acetone, anhydrous potassium carbonate was added, and the mixture was stirred for 10 min. Then 3-bromopropene was added. After the addition was complete, the mixture was heated to 50 °C and refluxed for 6-8 h. After the reaction was completed, the reaction solution was poured into ice water, and the pH was adjusted to 6-7 with 1 M hydrochloric acid solution. The mixture was extracted three times with ethyl acetate, and the ethyl acetate layers were combined. The mixture was washed with saturated brine, dried with anhydrous sodium sulfate, and purified by PTLC (developing solvent: dichloromethane:methanol = 12:1) to obtain modified icariin II. S2. Slowly add PBS solution to the purslane-derived vesicles and stir until homogeneous to obtain the purslane-derived vesicle solution. Dissolve the modified icariin II in DMSO and add it to the purslane-derived vesicle solution. Incubate at 37°C and 500 r / min for 6-8 h with shaking. Add the solution to a 100 kDa ultrafiltration tube and centrifuge at 4°C and 8000 rpm for 20 min to remove free modified icariin II, thus obtaining the icariin II complex preparation that protects against PC12 cell damage.

[0010] Further, in step S1, the molar concentration of icariin II in acetone is 0.02 mmol / mL.

[0011] Further, in step S1, the molar ratio of icariin II, anhydrous potassium carbonate, and 3-bromopropene is 2:3:3.

[0012] Furthermore, in step S2, the mass concentration of the purslane-derived vesicles in PBS is 500 μg / mL.

[0013] Further, in step S2, the mass ratio of the modified epimedium glycoside II to the purslane-derived vesicles is 1:15-20.

[0014] Further, in step S2, the mass concentration of the modified icariin II in DMSO is 200 μg / mL.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a compound formulation of icariin II to protect PC12 cells from damage. It combines chemically modified icariin II (modified icariin II) with naturally derived purslane vesicles, exhibiting excellent protective effects against hypoxic / glucose-deficient PC12 cells. A synergistic effect exists between the modified ICS II and the purslane vesicles. The purslane vesicles not only serve as a drug carrier but may also enhance the neuroprotective efficacy of modified icariin II through their own bioactive components. As a natural nanocarrier, purslane vesicles possess good biocompatibility and low immunogenicity. The successful loading of modified ICS II into vesicles helps protect it from in vivo degradation, prolonging its circulation time. Furthermore, the natural tropism of vesicles enhances drug accumulation at the site of nerve damage, achieving more efficient targeted delivery. This invention chemically modifies icariin II by introducing allyl groups, improving the lipid solubility and membrane permeability of the original ICS II, making it easier for cells to take up or interact with biological membranes, thereby enhancing its biological activity. This invention is the first to combine plant-derived vesicles with modified icariin II for the protection against nerve cell damage. The core ingredient of this invention, icariin II, is derived from the traditional Chinese medicine Epimedium, and the purslane-derived vesicles are obtained from the medicinal and edible plant Purslane. Using natural vesicles as a carrier offers better biodegradability and is expected to ensure safety. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only for this invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a scanning electron microscope image of the composite formulation described in Example 1 of the present invention; Figure 2 The particle size distribution of the composite formulation and purslane-derived vesicles described in Example 1 of this invention; Figure 3 This describes the protective effect of the composite formulations described in Examples 1-3 and Comparative Examples 1-3 of the present invention on PC12 cells. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. However, this invention is not limited to the following embodiments. It should be noted that, unless otherwise specified, all chemical reagents involved in this invention are purchased through commercial channels.

[0019] Example 1: An icariin II compound preparation for protecting PC12 cell damage, the raw materials for preparation include the following components: modified icariin II and purslane-derived vesicles.

[0020] The method for preparing purslane-derived vesicles includes the following steps: L1. After cleaning the purslane, cut it into small pieces and put it into a juicer. Add pre-cooled PBS solution to cover the purslane. Juice at 4°C for 1 min, stop for 1 min, and repeat 5 times. Filter the purslane juice with gauze to remove the residue. Take the filtrate and centrifuge at 1000×g for 10 min at 4°C. Take the supernatant and centrifuge at 3000×g for 20 min. Continue to take the supernatant and centrifuge at 10000×g for 30 min. Collect the supernatant. L2. Take the supernatant obtained in step L1, filter it through a 0.22 μm filter membrane, centrifuge at 100,000 × g for 2 h at 4 °C, resuspend the precipitate in PBS solution to obtain a resuspended solution, transfer the resuspended solution to a sucrose gradient solution (8%, 30%, 45% and 60%, dissolved in 20 mM Tris-Cl at pH 7.2), centrifuge at 150,000 × g for 2 h at 4 °C, collect the 45-60% band-like portion, wash it 3 times with PBS solution, resuspend it in PBS solution, freeze dry it to obtain purslane-derived vesicles.

[0021] This embodiment also provides a method for preparing the icariin II complex preparation for protecting PC12 cell damage, including the following steps: S1. Dissolve 0.06 mmol of icariin II in 3 mL of acetone, add 0.09 mmol of anhydrous potassium carbonate, stir for 10 min, then add 0.09 mmol of 3-bromopropene. After the addition is complete, reflux at 50 °C for 8 h. After the reaction is complete, pour the reaction solution into ice water, adjust the pH to 7 with 1 M hydrochloric acid solution, extract three times with ethyl acetate, combine the ethyl acetate layers, wash with saturated brine, dry with anhydrous sodium sulfate, and then purify by PTLC (developing solvent: dichloromethane:methanol = 12:1) to obtain modified icariin II. S2. Slowly add PBS solution to the purslane-derived vesicles and stir until homogeneous to obtain a purslane-derived vesicle solution of 500 μg / mL. Dissolve modified icariin II in DMSO to obtain a concentration of 200 μg / mL of modified icariin II in DMSO. Add the solution to the purslane-derived vesicle solution at a mass ratio of 1:20. Incubate at 37℃ and 500 r / min for 8 h with shaking. Add the solution to a 100 kDa ultrafiltration tube and centrifuge at 4℃ and 8000 rpm for 20 min to remove free modified icariin II, thus obtaining an icariin II complex preparation for protecting PC12 cells from damage.

[0022] Example 2: An icariin II compound preparation for protecting PC12 cell damage, the raw materials for preparation include the following components: modified icariin II and purslane-derived vesicles.

[0023] The method for preparing purslane-derived vesicles includes the following steps: L1. After cleaning the purslane, cut it into small pieces and put it into a juicer. Add pre-cooled PBS solution to cover the purslane. Juice at 4°C for 1 min, stop for 1 min, and repeat 5 times. Filter the purslane juice with gauze to remove the residue. Take the filtrate and centrifuge at 1000×g for 10 min at 4°C. Take the supernatant and centrifuge at 3000×g for 20 min. Continue to take the supernatant and centrifuge at 10000×g for 30 min. Collect the supernatant. L2. Take the supernatant obtained in step L1, filter it through a 0.22 μm filter membrane, centrifuge at 100,000 × g for 1.5 h at 4 °C, resuspend the precipitate in PBS solution to obtain a resuspended solution, transfer the resuspended solution to a sucrose gradient solution (8%, 30%, 45% and 60%, dissolved in 20 mM Tris-Cl at pH 7.2), centrifuge at 150,000 × g for 2 h at 4 °C, collect the 45-60% band-like portion, wash it 3 times with PBS solution, resuspend it in PBS solution, freeze dry it to obtain purslane-derived vesicles.

[0024] This embodiment also provides a method for preparing the icariin II complex preparation for protecting PC12 cell damage, including the following steps: S1. Dissolve 0.06 mmol of icariin II in 3 mL of acetone, add 0.09 mmol of anhydrous potassium carbonate, stir for 10 min, then add 0.09 mmol of 3-bromopropene. After the addition is complete, reflux at 50 °C for 6 h. After the reaction is complete, pour the reaction solution into ice water, adjust the pH to 6 with 1 M hydrochloric acid solution, extract three times with ethyl acetate, combine the ethyl acetate layers, wash with saturated brine, dry with anhydrous sodium sulfate, and then purify by PTLC (developing solvent: dichloromethane:methanol = 12:1) to obtain modified icariin II. S2. Slowly add PBS solution to the purslane-derived vesicles and stir until homogeneous to obtain a purslane-derived vesicle solution of 500 μg / mL. Dissolve modified icariin II in DMSO to obtain a concentration of 200 μg / mL. Add the solution to the purslane-derived vesicles at a mass ratio of 1:15. Incubate at 37℃ and 500 r / min for 6 h with shaking. Add the solution to a 100 kDa ultrafiltration tube and centrifuge at 4℃ and 8000 rpm for 20 min to remove free modified icariin II, thus obtaining an icariin II complex preparation for protecting PC12 cells from damage.

[0025] Example 3: An icariin II complex preparation for protecting PC12 cell damage, the raw materials for preparation include the following components: modified icariin II and purslane-derived vesicles.

[0026] The method for preparing purslane-derived vesicles includes the following steps: L1. After cleaning the purslane, cut it into small pieces and put it into a juicer. Add pre-cooled PBS solution to cover the purslane. Juice at 4°C for 1 min, stop for 1 min, and repeat 5 times. Filter the purslane juice with gauze to remove the residue. Take the filtrate and centrifuge at 1000×g for 10 min at 4°C. Take the supernatant and centrifuge at 3000×g for 20 min. Continue to take the supernatant and centrifuge at 10000×g for 30 min. Collect the supernatant. L2. Take the supernatant obtained in step L1, filter it through a 0.22 μm filter membrane, centrifuge at 100,000 × g for 1.8 h at 4 °C, resuspend the precipitate in PBS solution, transfer the resuspended solution to a sucrose gradient solution (8%, 30%, 45% and 60%, dissolved in 20 mM Tris-Cl at pH 7.2), centrifuge at 150,000 × g for 2 h at 4 °C, collect the 45-60% band-like portion, wash it three times with PBS solution, resuspend it in PBS solution, freeze dry it to obtain purslane-derived vesicles.

[0027] This embodiment also provides a method for preparing the icariin II complex preparation for protecting PC12 cell damage, including the following steps: S1. Dissolve 0.06 mmol of icariin II in 3 mL of acetone, add 0.09 mmol of anhydrous potassium carbonate, stir for 10 min, then add 0.09 mmol of 3-bromopropene. After the addition is complete, reflux at 50 °C for 7 h. After the reaction is complete, pour the reaction solution into ice water, adjust the pH to 6.5 with 1 M hydrochloric acid solution, extract three times with ethyl acetate, combine the ethyl acetate layers, wash with saturated brine, dry with anhydrous sodium sulfate, and then purify by PTLC (developing solvent: dichloromethane:methanol = 12:1) to obtain modified icariin II. S2. Slowly add PBS solution to the purslane-derived vesicles and stir until homogeneous to obtain a purslane-derived vesicle solution of 500 μg / mL. Dissolve modified icariin II in DMSO to obtain a concentration of 200 μg / mL. Add the solution to the purslane-derived vesicle solution at a mass ratio of 1:18. Incubate at 37℃ and 500 r / min for 7 h with shaking. Add the solution to a 100 kDa ultrafiltration tube and centrifuge at 4℃ and 8000 rpm for 20 min to remove free modified icariin II, thus obtaining an icariin II complex preparation for protecting PC12 cells from damage.

[0028] The only difference between Comparative Example 1 and Example 1 is that purslane-derived vesicles were used instead of the icariin II complex preparation used to protect PC12 cells from damage.

[0029] The only difference between Comparative Example 2 and Example 1 is that modified icariin II was used instead of the icariin II complex preparation that protects against PC12 cell damage.

[0030] The only difference between Comparative Example 3 and Example 1 is that icariin II was used instead of modified icariin II.

[0031] Experimental Example 1: The morphology of the purslane-derived vesicles prepared in Example 1 and the icariin II compound preparation for protecting PC12 cell damage, i.e., purslane-derived vesicles loaded with drug-modified icariin II, was observed using transmission electron microscopy. The results are as follows: Figure 1 As shown. The particle size distribution was analyzed, and the results are as follows. Figure 2 As shown.

[0032] Figure 1 The results showed that, compared with the blank purslane-derived vesicles, the membrane structure of the purslane-derived vesicles loaded with drug-modified icariin II (composite preparation) was still clearly visible, and the loading of drug-modified icariin II did not destroy the original morphology of the purslane-derived vesicles. Figure 2The results showed that, through particle size distribution analysis, the particle size of the drug-loaded purslane-derived vesicles (compound preparation) was slightly larger than that of the purslane-derived vesicles, which may be related to the loading of modified icariin II.

[0033] Experiment 2: Highly differentiated PC12 cells were seeded in DMEM high-glucose medium containing a mixture of 10% fetal bovine serum, 5% penicillin, and streptomycin (100×) and incubated at 37°C in a 5% CO2 incubator, passaged once every 48 h. The experimental groups were: blank group, control group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group. PC12 cells in the logarithmic growth phase were collected, and the cell suspension density was adjusted to 1.5 × 10⁻⁶. 5 Cells / mL were seeded in 96-well plates and cultured for 24 h, then the original culture medium was discarded. The blank group was washed twice with sugar-containing EBSS, then sugar-containing EBSS was added and the plates were incubated at 37°C and 5% CO2 for 24 h. The control group, Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were washed twice with sugar-free EBSS, sugar-free EBSS was added, and the plates were incubated at 37°C for 2 h in a closed hypoxic chamber with 5% CO2 and 95% N2. The control group was replaced with complete culture medium, while Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were replaced with complete culture medium containing the compound preparations prepared in Examples 1-3 and Comparative Example 1-3, respectively. The dosage was calculated as 25 μM of icariin II or modified icariin II. The plates were incubated at 37°C and 5% CO2 for 24 h. Add 20 μL of 5 g / L MTT to each well and incubate at 37℃ in a 5% CO2 incubator in the dark for 4 h. Discard the supernatant, add 120 μL of DMSO, and shake gently for 5 min. Read the absorbance (OD) at 570 nm using a microplate reader. Calculate cell viability: Cell viability (%) = (OD treatment group / OD blank group) × 100%. Results are as follows: Figure 3 As shown.

[0034] This invention uses the MTT assay to detect PC12 cell viability. Figure 3 The results showed that the cell survival rate of the control group was significantly lower than that of the blank group, Examples 1-3 and Comparative Examples 1-3. The compound preparation of the present invention improved cell viability after action, and the compound preparation prepared by the present invention can effectively protect PC12 cells from damage.

[0035] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the invention is limited to these examples; within the framework of the invention, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.

Claims

1. A compound preparation of icariin II to protect PC12 cells from damage, characterized in that, The raw materials for preparation include the following components: modified icariin II and purslane-derived vesicles; The method for preparing the purslane-derived vesicles includes the following steps: L1. After cleaning the purslane, cut it into small pieces, put it into a juicer, add pre-cooled PBS solution to cover the purslane, juice it, filter the purslane juice with gauze, remove the filter residue, centrifuge the filtrate, and collect the supernatant. L2. Take the supernatant obtained in step L1, filter it through a 0.22 μm filter membrane, centrifuge it at high speed, resuspend the precipitate in PBS solution, perform sucrose gradient purification on the resuspended solution, collect 45-60% of the banded portion, wash it with PBS solution, resuspend it in PBS solution, freeze dry it to obtain purslane-derived vesicles.

2. The icariin II complex preparation for protecting PC12 cell damage according to claim 1, characterized in that, In step L1, the centrifugation is performed at 4°C, centrifuging at 1000×g for 10 min, taking the supernatant and centrifuging at 3000×g for 20 min, and then taking the supernatant again and centrifuging at 10000×g for 30 min.

3. The icariin II complex preparation for protecting PC12 cell damage according to claim 2, characterized in that, In step L2, the high-speed centrifugation is performed at 4°C for 1.5-2 hours at 100,000 × g.

4. The icariin II complex preparation for protecting PC12 cell damage according to claim 3, characterized in that, In step L2, the sucrose gradient purification involves transferring the resuspended solution to 8%, 30%, 45%, and 60% sucrose gradient solutions and centrifuging at 4°C and 150,000 × g for 2 h.

5. A method for preparing an icariin II complex preparation for protecting PC12 cell damage as described in any one of claims 1-4, characterized in that, Includes the following steps: S1. Icariin II was dissolved in acetone, anhydrous potassium carbonate was added, and the mixture was stirred. 3-bromopropene was added, and the mixture was refluxed. After the reaction was completed, the reaction solution was poured into ice water, and the pH was adjusted to 6-7 with 1 M hydrochloric acid solution. The mixture was extracted with ethyl acetate, the ethyl acetate layers were combined, washed, dried, and purified to obtain modified iccariin II. S2. Add PBS solution to purslane-derived vesicles, stir well to obtain purslane-derived vesicle solution, dissolve modified icariin II in DMSO, add to purslane-derived vesicle solution, shake and incubate, ultrafilter to obtain icariin II complex preparation for protecting PC12 cell damage.

6. The method for preparing the icariin II complex preparation for protecting PC12 cell damage according to claim 5, characterized in that, In step S1, the molar ratio of icariin II, anhydrous potassium carbonate, and 3-bromopropene is 2:3:

3.

7. The method for preparing the icariin II complex preparation for protecting PC12 cell damage according to claim 6, characterized in that, In step S2, the mass ratio of the modified epimedium glycoside II to the purslane-derived vesicles is 1:15-20.

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