Rheum officinale-salvia miltiorrhiza self-assembled nanoparticles and application thereof

Abstract

The invention discloses a rhubarb-salvia miltiorrhiza self-assembled nanoparticle and an application thereof. The self-assembled nanoparticles are separated from an ethanol extracting solution of a rhubarb and salvia miltiorrhiza compound by means of differential centrifugal impurity removal and the like. The average particle size of the nanoparticles is 500 nm, the Zeta potential is shown in the specification, the structural integrity of the nanoparticles is kept under the conditions that the temperature is-80 DEG C, the pH value is 2.2 and the pH value is 7.4, and the nanoparticles have good low-temperature stability and acid-base stability; a cisplatin-induced mouse acute kidney injury model shows that the rhubarb-salvia miltiorrhiza nano self-assembled particle has a good prevention effect on acute kidney injury, and can be widely applied to preparation of drugs for preventing acute kidney injury.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine nanomedicine, specifically to a rhubarb-salvia miltiorrhiza self-assembled nanoparticle and its application in the prevention of acute kidney injury. Background Technology

[0002] Acute kidney injury (AKI) is a critical clinical condition, and current treatments are mainly symptomatic and supportive, lacking specific repair drugs. While existing rhubarb and tanshinone preparations have clear renal protective effects, their active ingredients have low bioavailability and lack renal targeting. Current nanodelivery technologies for traditional Chinese medicine mostly rely on exogenous carrier materials, posing safety risks and negating the synergistic advantages of traditional Chinese medicine formulations. Although self-assembly strategies have been explored in the field of traditional Chinese medicine, there is a lack of research on the co-assembly of rhein and tanshinone through intermolecular forces to construct carrier-free, renal-targeting nanodelivery systems; this technology remains a gap in the field. Summary of the Invention

[0003] The purpose of this invention is to address the aforementioned problems in the existing technology and provide rhubarb-salvia miltiorrhiza self-assembled nanoparticles and their application in the prevention of acute kidney injury. Based on the intermolecular hydrogen bonds and π-π stacking interactions of the active components of rhubarb-salvia miltiorrhiza, this invention separates high-purity self-assembled carrier-free nanoparticles from the ethanol extract of rhubarb-salvia miltiorrhiza using a simple centrifugation process. This achieves the co-delivery and synergistic effect of the active components of the traditional Chinese medicine compound, improving its stability and absorption efficiency, thereby enhancing its preventive effect against acute kidney injury.

[0004] The technical solution adopted in this invention is as follows:

[0005] A rhubarb-salvia miltiorrhiza self-assembled nanoparticle is prepared by concentration and differential centrifugation of an ethanol extract of a rhubarb-salvia miltiorrhiza compound; the nanoparticles are assembled via hydrogen bonds and... Formed through self-assembly via stacking, with an average particle size of 500 nm and a Zeta potential of [missing value]. Furthermore, the nanoparticle structure remains stable under conditions of -80℃ and pH 2.2 and pH 7.4.

[0006] Preferably, the method for preparing the nanoparticles includes the following steps:

[0007] S1. Weigh 20 g of rhubarb and salvia miltiorrhiza slices respectively in a 1:1 ratio, pulverize them and pass them through a 40-mesh sieve to obtain a uniform powder; add 10 times the volume of 70% ethanol, soak at room temperature for 1 h, then reflux for 3 h and filter; repeat the extraction with 70% ethanol once more, filter, combine the two filtrates, mix them and concentrate under reduced pressure using a rotary evaporator to obtain a rhubarb-salvia miltiorrhiza extract (RSE) containing 0.2 g crude drug / mL;

[0008] S2. Centrifuge RSE at 3000 rpm for 10 min to remove larger particles from the solution, and then take the supernatant and centrifuge at 15000 rpm at 4°C for 40 min. After centrifugation, discard the supernatant, redisperse the precipitate in deionized water, and repeat the centrifugation-resuspending cycle three times to remove residual impurities. Finally, filter the suspension through a 1 μm needle filter to remove potential aggregates, and freeze-dry to obtain rhubarb-salvia miltiorrhiza self-assembled nanoparticles RSNPs.

[0009] Preferably, in step S2, the freeze-drying is performed at -50°C and 0.01 MPa for 72 hours.

[0010] This invention also provides the application of the above-mentioned rhubarb-salvia miltiorrhiza self-assembled nanoparticles in the preparation of drugs for the prevention of acute kidney injury.

[0011] The beneficial effects of this invention are as follows:

[0012] 1. This invention separates self-assembled nanoparticles from the ethanol extract of rhubarb and salvia miltiorrhiza compound using low-speed and low-temperature high-speed centrifugation to remove impurities. These nanoparticles have an average particle size of 500 nm, a Zeta potential of -27.59 ± 6.19 mV, and maintain their structural integrity at -80℃ and under pH 2.2 and pH 7.4 conditions, exhibiting low-temperature stability and good acid-base stability.

[0013] 2. The rhubarb-Salvia miltiorrhiza self-assembled nanoparticles of this invention were identified by UPLC-MS compositional analysis as containing 101 different phytochemicals (RSNPs). Using a cisplatin-induced mouse model of acute kidney injury, the rhubarb-Salvia miltiorrhiza self-assembled nanoparticles demonstrated a good preventive effect against acute kidney injury, significantly reducing serum urea nitrogen (BUN) and creatinine (Scr) levels, and significantly reducing renal levels of glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), and interleukin. Interleukin-6 (IL-6) and tumor necrosis factor Its content allows for wide application in the preparation of drugs for the prevention of acute kidney injury. Attached Figure Description

[0014] Figure 1 This refers to the preparation of RSNPs, a traditional Chinese medicine compound nanoparticle, in Example 1.

[0015] Figure 2 The chromatogram of positive ion BPIs for RSNPs in Example 2;

[0016] Figure 3 The negative ion BPI chromatogram of RSNPs in Example 2;

[0017] Figure 4 This is the particle size distribution map of RSNPs measured by dynamic light scattering in Example 2;

[0018] Figure 5 The image shows the Zeta potential diagram of RSNPs obtained by phase analysis light scattering in Example 2.

[0019] Figure 6 These are transmission electron microscopy (TEM) images of the RSNPs from Example 2.

[0020] Figure 7 The Fourier transform infrared spectrum of RSNPs in Example 2;

[0021] Figure 8 The image shows the UV absorption spectrum of the RSNPs in Example 2.

[0022] Figure 9 The particle size changes of RSNPs in Example 3 after storage at -80°C for different time periods;

[0023] Figure 10 The particle size variation of RSNPs under different pH conditions in Example 3;

[0024] Figure 11 To evaluate the intervention effect of RSNPs on the acute kidney injury model in mice using the appearance (A) and kidney index (B) of the mice in Example 4;

[0025] Figure 12 To evaluate the intervention effect of RSNPs on the acute kidney injury model using blood urea nitrogen concentration (A) and serum creatinine concentration (B) in Example 4;

[0026] Figure 13 To assess the intervention effect of RSNPs on an acute kidney injury model in Example 4, the renal glutathione concentration was evaluated.

[0027] Figure 14 To assess the intervention effect of RSNPs on an acute kidney injury model in Example 4, the renal catalase concentration was evaluated.

[0028] Figure 15 To assess the intervention effect of RSNPs on an acute kidney injury model in Example 4, the renal superoxide dismutase concentration was evaluated.

[0029] Figure 16 To assess the intervention effect of RSNPs on an acute kidney injury model in Example 4, the renal interleukin-1β concentration was evaluated.

[0030] Figure 17 To assess the intervention effect of RSNPs on an acute kidney injury model in Example 4, the renal interleukin-6 concentration was evaluated.

[0031] Figure 18 To assess the intervention effect of RSNPs on an acute kidney injury model in Example 4, the concentration of renal tumor necrosis factor-α was evaluated. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0033] Example 1: Preparation of RSNP nanoparticles in traditional Chinese medicine compound prescriptions

[0034] Preparation route of nano-Chinese medicine compound as follows Figure 1 As shown, the specific preparation method includes: weighing 20 g of rhubarb and salvia miltiorrhiza slices in a 1:1 ratio, pulverizing them, and passing them through a 40-mesh sieve to obtain a uniform powder. Add 10 times the volume of 70% ethanol, soak at room temperature for 1 h, then reflux for 3 h, and filter. Repeat the extraction with 70% ethanol once more, filter, combine the two filtrates, mix them, and concentrate under reduced pressure using a rotary evaporator to obtain a solution containing 0.2 g / mL of rhubarb-salvia miltiorrhiza extract (RSE).

[0035] Self-assembled nanoparticles in rhubarb-Salvia miltiorrhiza (RSE) were separated using differential centrifugation. First, the RSE was centrifuged at 3000 rpm for 10 min to remove larger particles. Then, the supernatant was centrifuged at 15000 rpm at 4°C for 40 min. After centrifugation, the supernatant was discarded, and the precipitate was redispersed in deionized water. This centrifugation-resuspending cycle was repeated three times to remove residual impurities. Finally, the suspension was filtered through a 1 μm needle filter to remove potential aggregates. After freeze-drying, the self-assembled rhubarb-Salvia miltiorrhiza nanoparticles, termed RSNPs, were obtained.

[0036] Example 2: Compositional Analysis and Characterization of RSNPs

[0037] This embodiment performs component analysis and characterization on the rhubarb-salvia miltiorrhiza self-assembled nanoparticles (RSNPs) prepared in Example 1. Figures 2-8 ).

[0038] ① Component analysis of RSNPs by UPLC-MS / MS

[0039] UPLC-MS analysis identified 101 different phytochemicals within the RSNPs. The detection pattern distribution showed that 63 compounds ionized in positive ion mode and 38 in negative ion mode. Structural classification categorized these compounds into five major chemical classes: alkaloids and their derivatives, lipids and lipid molecules, phenylpropanoids and polyketides, organic acids and their derivatives, and benzene ring compounds.

[0040] ② Determination of particle size, PDI, and Zeta potential of RSNPs

[0041] After diluting RSNPs by a certain factor, the particle size distribution, polydispersity index (PDI), and zeta potential were measured using a Malvern laser particle size analyzer. Each sample was measured three times. The results showed that the prepared RSNPs had an average particle size of approximately 500 nm and a relatively uniform particle distribution (PDI = 0.278). Simultaneously, the zeta potential of the nanoparticles was... This further reflects the stability and uniformity of RSNPs.

[0042] ③ Transmission electron microscopy examination of RSNPs

[0043] RSNPs were dropped onto the surface of a copper mesh coated with a carbon film. After standing for 3 minutes, excess liquid was absorbed with filter paper, and then 10 μL of phosphotungstic acid solution (2%, w / v) was added for staining for 1 minute. After air drying, the mesh was imaged under an accelerating voltage of 120 kV. The results showed that under TEM, the RSNPs were spherical and relatively uniform in size.

[0044] ④ Fourier transform infrared spectroscopy scan

[0045] Weigh appropriate amounts of RSNPs and RSE lyophilized powder, mix them separately with dry KBr fine powder, grind and mix evenly, then compress into uniform thin tablets using a tablet press. Fourier transform infrared spectroscopy was used to analyze the samples at 400–4000 cm⁻¹. -1 The infrared absorption spectra of RSNPs were measured in the wavenumber range. The results showed that RSNPs were at 3424 cm⁻¹. -1 The presence of stretching vibration absorption peaks of OH groups at the RSE indicates enhanced hydrogen bonding, suggesting that hydrogen bonding may be a key factor in the formation of rhubarb-salvia miltiorrhiza self-assembled nanoparticles.

[0046] ⑤ Ultraviolet-Visible Absorption Spectroscopy Analysis

[0047] The UV-Vis absorption spectra were measured using a UV-3600 Plus UV spectrophotometer. RSNPs and RSE solutions were placed in a 1 cm path length quartz cuvette, and the wavelength range of 200–800 nm was scanned, with deionized water as a blank reference. The results showed that compared to RSE, the RSNPs spectrum exhibited a decrease in absorption intensity at 220 nm accompanied by a blue shift, which is consistent with… This is consistent with the stacking-driven H-aggregation behavior, indicating that... Stacking may be a key factor promoting the formation of rhubarb-salvia miltiorrhiza self-assembled nanoparticles.

[0048] Example 3: Stability of RSNPs

[0049] Low-temperature storage stability: The freeze-dried RSNPs (prepared in Example 1) powder was stored at -80℃ for 1, 3, and 5 days, and samples were taken at each time point for reconstitution and particle size distribution was measured. Acid-base stability: RSNPs were dispersed in buffer systems at pH 2.2 and pH 7.4, and incubated at 37℃ for a certain period of time. Changes in particle size distribution and aggregation state were then measured. The results showed that RSNPs maintained structural integrity under physiologically relevant pH conditions (pH 2.2 and pH 7.4), indicating strong tolerance to the gastrointestinal environment and highlighting their potential for oral administration. Figure 10 Meanwhile, stability tests under refrigeration conditions further corroborated the stability of this nano-formulation. Figure 9 ).

[0050] Example 4: Evaluation of the preventive effect of RSNPs on an acute kidney injury model

[0051] To evaluate the biocompatibility of RSNPs (prepared in Example 1) and their preventive effect on a mouse model of acute kidney injury, ICR mice were randomly divided into 5 groups (n = 10 per group): control group (saline), model group (cisplatin), RSE group (RSE 1.0 g / kg + cisplatin), low-dose RSNPs group (0.5 g / kg RSNPs + cisplatin), and high-dose RSNPs group (1.0 g / kg RSNPs + cisplatin). Mice in the drug groups were administered the drug via gavage for 7 consecutive days, once daily. The control and model groups were administered saline via gavage for 7 consecutive days instead of the drug. Except for the control group, all other groups received a single intraperitoneal injection of cisplatin (15 mg / kg) 1 hour after drug administration on day 4 to induce acute kidney injury in mice. Mice were fasted for 12 hours after the last administration, but allowed free access to water. Blood and kidney samples were collected after anesthesia. The effects of RSNPs were evaluated by measuring serum urea nitrogen (BUN) and creatinine (Scr) levels, and by measuring renal glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). Figures 11-18 ).

[0052] Gross anatomical observations showed that RSNPs (especially in the high-dose group) significantly alleviated cisplatin-induced renal enlargement and pallor, and that RSNPs dose-dependently reduced elevated renal indices, normalized serum creatinine and blood urea nitrogen levels, and protected renal function. Simultaneously, RSNPs dose-dependently restored the activity of key antioxidant enzymes (GSH, CAT, SOD) and reversed GSH depletion, maintaining renal redox homeostasis. Furthermore, RSNPs inhibited cisplatin-induced elevation of pro-inflammatory mediators (TNF-α, IL-1β, IL-6), suppressing renal inflammatory responses. These results indicate that prophylactic administration of RSNPs can provide multidimensional protection against cisplatin nephrotoxicity by synergistically maintaining redox balance, regulating inflammatory responses, and protecting renal structure.

[0053] The specification and drawings of this invention are intended to be illustrative rather than restrictive. Based on this invention, those skilled in the art can make substitutions and modifications to some of the technical features without creative effort, and all such modifications are within the scope of protection of this invention.

Claims

1. A rhubarb-salvia miltiorrhiza self-assembled nanoparticle, characterized in that, The nanoparticles were prepared by concentration and differential centrifugation of the ethanol extract of rhubarb-salvia miltiorrhiza compound; the nanoparticles are obtained through hydrogen bonding and... Formed through self-assembly via stacking, with an average particle size of 500 nm and a Zeta potential of [missing value]. Furthermore, the nanoparticle structure remains stable under conditions of -80℃ and pH 2.2 and pH 7.

4.

2. The rhubarb-Salvia miltiorrhiza self-assembled nanoparticles according to claim 1, characterized in that, The preparation method of the nanoparticles includes the following steps: S1. Weigh 20 g of rhubarb and salvia miltiorrhiza slices respectively in a 1:1 ratio, pulverize them and pass them through a 40-mesh sieve to obtain a uniform powder; add 10 times the volume of 70% ethanol, soak at room temperature for 1 h, then reflux for 3 h and filter; repeat the extraction with 70% ethanol once more, filter, combine the two filtrates, mix them and concentrate under reduced pressure using a rotary evaporator to obtain a rhubarb-salvia miltiorrhiza extract (RSE) containing 0.2 g crude drug / mL; S2. Centrifuge RSE at 3000 rpm for 10 min to remove larger particles from the solution, and then take the supernatant and centrifuge at 15000 rpm at 4°C for 40 min. After centrifugation, discard the supernatant, redisperse the precipitate in deionized water, and repeat the centrifugation-resuspending cycle three times to remove residual impurities. Finally, filter the suspension through a 1 μm needle filter to remove potential aggregates, and freeze-dry to obtain rhubarb-salvia miltiorrhiza self-assembled nanoparticles RSNPs.

3. The rhubarb-Salvia miltiorrhiza self-assembled nanoparticles according to claim 2, characterized in that, In S2, freeze drying is performed at -50°C and 0.01 MPa for 72 h.

4. The application of rhubarb-salvia miltiorrhiza self-assembled nanoparticles as described in any one of claims 1-3 in the prevention of acute kidney injury.

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