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Back to Journal »International Journal of Nanomedicine» Volume 16

The natural nano drug delivery system in Coptis extract and the pharmacokinetics of modified berberine hydrochloride

Authors: Zhao Jie, Zhao Q, Lu Jinzhong, Ye De, Mu Shi, Yang Xinde, Zhang Weide, Ma BL

Published on September 14, 2021, the 2021 volume: 16 pages 6297—6311

DOI https://doi.org/10.2147/IJN.S323685

Single anonymous peer review

Editor approved for publication: Dr. Ebrahim Mostafavi

Zhao Jing,1,* Zhao Qing,1,* Lu Jingze,1 Ye Dan,1 Shengmu,1 Yang Xiaodi,2 Zhang Weidong,3,4 Ma Bingliang1 1 Department of Pharmacology, Shanghai University, Shanghai, 201203; 2 Shanghai Traditional Chinese Medicine University Institute of Chinese Medicine Innovation, Shanghai 201203; 3 Interdisciplinary Institute of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 201203; 4 School of Pharmacy, Second Military Medical University, Shanghai, 200433 *The above authors have equal contributions to this article Bing-Liang Ma Electronics Mail [email protected]; [email protected] Purpose: This study aims to evaluate the effects of natural nanoparticles (Nnps) isolated from Coptis extract on the drug and pharmacokinetics of berberine hydrochloride (BBR), and systematically To explore relevant mechanisms. Method: Firstly, Nnps was separated from the extract of Coptis chinensis, and then the Nnps-BBR complex was prepared. Through qualitative and quantitative analysis of the size, Zeta potential, morphology and composition of Nnps and Nnps-BBR complexes, the effect of Nnps on BBR crystallization was characterized. Then the effect of Nnps on the solubility and solubility of BBR was evaluated. In addition, the effects of Nnps on BBR in mouse cell uptake, transmembrane transport, metabolic stability and pharmacokinetics were studied. Results: The average size of Nnps is 166.6 ± 1.3 nm, and the Zeta potential is − 12.5 ± 0.2 mV. Nnps is formed by the denaturation of coexisting plant proteins with a molecular weight of <30 kDa. Nnps adsorbs or disperses BBR, which promotes the transformation of BBR from crystal to amorphous form, and improves its solubility and solubility. Nnps carries and promotes the uptake of BBR by human colon adenocarcinoma (Caco-2) cells through pit-mediated endocytosis, reduces P-gp-mediated BBR in the mouse intestinal sac and stably expresses the transporter P-gp Outflow (MDCK-MDR1) cells in Madin-Darby canine kidney cells. In addition, Nnps improved the metabolic stability of BBR in mouse intestinal S9 and promoted the intestinal absorption of BBR in mice, such as peak BBR concentration (Cmax, 1182.3 vs. 310.2 ng/mL) and exposure level (AUC0-12 hours, The increase of 2842.8 to 1447.0 ng) is shown in the mouse portal vein. In addition, Nnps increased the exposure level of BBR in the liver of mice (95,443.2 vs 43,586.2 ng·h/g liver). Conclusion: The protein nanoparticles isolated from Coptis extract can form a natural nano-drug system with BBR, thereby significantly improving the pharmacokinetics of oral BBR. Keywords: natural nanoparticles, drug delivery system, pharmacokinetic synergy, berberine hydrochloride, herbal extract

Herbal extracts are an important part of traditional medicine and are widely used worldwide. 1 In addition, herbal extracts are an important source of new drugs because they contain a variety of compounds with novel structures and significant biological activities. 2 Unfortunately, many compounds purified from herbal extracts are difficult to absorb after oral administration, which severely limits their clinical applications. For example, the oral bioavailability of the active ingredients of some representative traditional Chinese medicines (TCMs), such as Tanshinone IIA, 3 Berberine Hydrochloride (BBR), 4 Astragaloside IV, 5 Baicalin, 6 Curcumin, 7 Ginsenoside Rb1, Rb2, Rb3, 8 glycyrrhizin, 9 and platycodin D10 are all less than 5%. However, the absorption of these compounds is usually better after oral administration of the corresponding herbal extracts. 11 In the case of tanshinone IIA, the exposure level of tanshinone IIA in rats that received oral salvia extract was more than 19 times higher. In rats receiving oral tanshinone IIA. 3

The pharmacokinetic synergy occurs in herbal extracts. 11 For example, some secondary metabolites contribute to synergy. 11 More importantly, there are some primary metabolites in herbal extracts, including proteins and polysaccharides, which are usually high in content. The role of primary metabolites in herbal extracts has attracted more and more attention. 11 For example, some polysaccharides have prebiotic-like effects, which can increase the hydrolysis of saponins by promoting the proliferation of intestinal bacteria and improving their metabolic capacity, thereby producing glycoside ligands. 12,13 More importantly, proteins and polysaccharides can directly change the activity The current form of the ingredient and intestinal absorption. In Zhuang’s research, natural nanoparticles (Nnps) were observed in water extracts of 60 Chinese medicines and 24 Chinese medicine formulations. 14 Nnps is usually composed of proteins, polysaccharides and lipids. 15-19 Importantly, Nnps can adsorb active ingredients, 17, 20-22 carry them for absorption, 20, 23, 24 and distribute them to target tissues. 17 These results indicate that Nnps may affect the pharmaceutical and pharmacokinetic properties of active ingredients in herbal extracts. However, no systematic research has been conducted so far.

Coptis chinensis is the dried rhizome of Coptis chinensis, CY Chen et Hsiao or Coptis chinensis. It is one of the most commonly used Chinese medicines. BBR has a wide range of pharmacological effects and is the main active component of Coptidis. 25 But after oral administration, due to poor water solubility, about 56% of BBR is directly excreted in feces. 4,26,27 In addition, BBR is excreted by intestinal efflux transporters such as p-glycoprotein (P-gp) and metabolized by intestinal drug-metabolizing enzymes, resulting in 43.5% of the oral dose being eliminated by the intestine. 4,26,27 After entering the liver 4,26,27 In short, due to poor solubility and significant first pass elimination, the bioavailability of oral BBR is as low as 0.36%. 4,26,27 However, it was found that the exposure level of BBR in mice that took Coptidis extract orally may be 15 times higher than that of mice that took Coptidis extract orally. 20,28 In addition, Nnps are formed spontaneously from the proteins in Coptis extract, and these Nnps can adsorb and carry y BBR to be absorbed by intestinal epithelial cells. 20 However, the effects and mechanisms of these Nnps on the drug and pharmacokinetic properties of BBR remain to be elucidated.

Therefore, this study aims to evaluate the pharmacological and pharmacokinetic effects of Nnps isolated from Coptis extracts on BBR, and to systematically explore the relevant mechanisms. In view of the widespread existence of Nnps in herbal extracts, this research is expected to promote more research on the interaction of Nnps with small molecular components in herbal extracts.

The dried coptis was purchased from Shanghai Kangqiao Chinese Medicine Decoction Pieces Co., Ltd. (Shanghai, China). These herbal tablets were collected and produced in Sichuan Province, China. According to the "People's Republic of China Pharmacopoeia" (2015 edition), this Chinese herbal medicine piece was identified as the root of C. chinensis. The voucher specimen (No.180305) is deposited in the Department of Pharmacology, School of Pharmacy, Shanghai University of Traditional Chinese Medicine. According to regional guidelines, no further research approval is required.

The purity of all reference compounds used in this study exceeds 98%. BBR, berberine, epiberberine, palmatine, norberberine, carbamazepine, verapamil hydrochloride, filter (0.22 μm) and dialysis membrane (3500 D) were purchased from Shanghai Yuanye Biological Co., Ltd. Company (Shanghai, China). Amiloride, cytochalasin D and indomethacin were purchased from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). Chlorpromazine, dimethyl sulfoxide and acetonitrile were purchased from Merck & Co., Inc. (New Jersey, USA). BCA (quinolinic acid) protein detection kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd. (Shanghai, China). The combined CD-1 mouse intestinal S9 fraction was obtained from SEKISUI Medical Co. Ltd (Tokyo, Japan). Formic acid, ammonium formate, fetal bovine serum and Dulbecco's Modified Eagle Medium (DMEM) are products of Thermo Fisher Scientific (Massachusetts, USA). Trypsin and penicillin-streptomycin solutions were purchased from Biosharp (Hefei, China). The pure water used in this study was prepared using the Millipore Milli-Q system (Massachusetts, USA).

Human colon adenocarcinoma cells (Caco-2) and Madin-Darby canine kidney cells stably expressing the transporter P-gp (MDCK-MDR1) were provided and verified by Sandia Pharmaceutical Technology (Shanghai) Co., Ltd. (Shanghai, China). The cells were cultured in DMEM supplemented with 10% FBS, penicillin-streptomycin solution and HEPES (15 mM) at 37°C in a humidified atmosphere of 5% CO2. Hank Balanced Salt Solution (HBSS) [composed of (mM) 135 NaCl, 1.2 MgCl2, 0.81 MgSO4, 27.8 glucose, 2.5 CaCl2 and 25 HEPES, pH 7.2)] is used to replace the medium in the incubation experiment. When dimethyl sulfoxide (DMSO) is used, its final concentration in HBSS is limited to less than 1‰.

ICR mice (Class II, male and female, body weight 24 ± 2 g) were purchased from Shanghai Slac Laboratory Animal Co., Ltd. (Shanghai, China). The mice were kept in an air-conditioned room at 22-24°C with a dark/light cycle of 12/12 hours. Before the experiment, the mice fasted for about 12 hours, but allowed to drink freely. All animal experiment protocols were approved by the Institutional Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine (PZSHUTCM19011105). All experiments were carried out in accordance with the guidelines for the care and use of laboratory animals in Shanghai University of Traditional Chinese Medicine.

The LC-MS system consists of a Shimadzu HPLC (LC-20AD) system (Kyoto, Japan) and a Thermo Scientific LCQ fleet mass spectrometer (Massachusetts, USA) equipped with an electrospray ionization (ESI) source. Eclipse XDB-C18 (4.6 × 150 mm, 5 µm) was used to keep at room temperature for chromatographic separation of analytes. Use water containing formic acid (0.0625%) and ammonium formate (4 mM) as mobile phase A, and methanol as mobile phase B. Use the following elution gradient with a flow rate of 0.3 mL/min: 0–7 minimum, 20% to 30% B; 7.01–10 minutes, 20–20% B. Data acquisition is performed in the selected ion monitoring mode. Berberine and epiberberine (m/z 336.2, retention time is different), berberine (m/z 320.2), palmatine (m/z 352.1) and carbamazepine (internal standard, m/z) Protonated [MH] ion 237.0) is generated. The linear dynamic range of berberine, epiberberine, berberine, and palmatine is 0.156 to 10 μg/mL. The method has been verified in terms of accuracy, precision, recovery, repeatability and stability (data not shown), and meets the requirements for quantitative analysis of alkaloids in samples with higher concentrations.

The biological samples in this study were precipitated in three times the volume of acetonitrile. After centrifugation at 4°C at 16,000 rpm for 10 min, add an equal volume of water and mix with the supernatant. Then, an LC-MS/MS system was used to inject 10 μL of each sample for analysis. The system consisted of an HPLC (LC-20AD) from Shimadzu and a Thermo Scientific TSQ Quantum Ultra mass spectrometer (Massachusetts, USA). C18 analytical column (Hypersil Gold, 5 µm, 100×2.1 mm) is used for chromatographic separation of analytes. The mobile phase consists of solvent A (aqueous solution of formic acid [0.08%, v/v] and ammonium acetate [2 mM]) and solvent B (acetonitrile). Use the following elution gradient with a flow rate of 0.3 mL/min: 0-7 min, 15% to 68% B; 7.01-10 min, 15-15% B. ESI source adopts positive ion mode, and data collection adopts multiple reaction monitoring mode ：M/z 336.2→322.3, m/z 324.3→308.0, m/z 237.0→194.3 are respectively berberine, norberberine and carbamazepine (internal standard). The linear range of berberine is 1.95–1000 ng/mL. Our published research uses a validated method that meets the requirements for quantitative analysis of alkaloids in biological samples with relatively low concentrations. 29,30

In short, the Huanglian herbal tablets were extracted twice with 10 times the volume of boiling water (1.5 hours for the first extraction and 1 hour for the second extraction). The resulting water extract was then filtered through eight layers of gauze and dried under vacuum at 60°C.

The content of berberine, epiberberine, berberine, palmatine and palmatine in the extract of coptis was detected for quality control. In short, the extract powder was dissolved in methanol (1 mg/mL) and sonicated for 60 minutes. After centrifugation at 16,000 rpm for 10 minutes, the supernatant was injected into the LC-MS system for quantitative analysis.

The Coptis extract powder is dissolved in water. After sonicating for 1 h, the solution was centrifuged at 3000 rpm for 10 min. The obtained supernatant was filtered through a 0.22-μm filter. The filtered solution was then dialyzed with water in a dialysis bag (3500 D) for five consecutive days. Nnps is obtained by freeze-drying the residue in the dialysis bag. In addition, Nnps and BBR are dissolved in water at a weight ratio of 1:1. The solution was then boiled for 1 hour, and then lyophilized to obtain Nnps-BBR complex powder.

A Malvern Zetasizer Nano analyzer (Worcestershire, UK) was used to determine the particle size and Zeta potential of the Nnps or Nnps-BBR complex aqueous solution. The powder of Nnps or Nnps-BBR composite was sprayed with gold, dried in vacuum, and then observed under a FEI Quanta 250 Scanning Electron Microscope (SEM) (Oregon, USA) at 10 kV. A Leica SP8 Confocal Laser Fluorescence Microscope (LCFM) (Wetzlar, Germany) was used to observe the morphology of Nnps or Nnps-BBR composite powder. Use the BCA kit to determine the protein content in Nnps. Using glucose as a reference standard, the content of polysaccharides in Nnps was determined by phenol-sulfuric acid method. The LC-MS method was used to determine the content of alkaloids in Nnps or Nnps-BBR complex.

In order to obtain undenatured Nnps forming protein, a new Coptis extract was prepared according to the method described in the section "Preparation and Quality Control of Coptis extract", but the extraction temperature was kept at 50°C. The protein in the extract was obtained by dialysis according to the method described in the section "Preparation and Characterization of Nnps and Nnps-BBR Complex". After dissolution, the purity and molecular weight of the protein were determined by standard gel electrophoresis (SDS-PAGE) in 4% concentrated gel and 10% separating gel and stained with silver using a protein gel electrophoresis device (Bio-Rad, USA). In addition, After the protein solution was boiled for 1 h, the particle size and Zeta potential of the formed particles were measured using the Malvern Zetasizer Nano analyzer.

The morphology of BBR, Nnps and Nnps-BBR complex was observed by SEM. Differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD) were also performed to verify the influence of Nnps on the crystal form of BBR. For DSC analysis, approximately 1.5 mg of powder sample was placed in an open aluminum crucible and heated from 20°C to 20°C at a rate of 10°C/min using TA DSC Q2000 Differential Scanning Calorimeter (Delaware, USA) 320°C. For PXRD analysis, a Bruker D2 phase shifter (Rheinstetten, Germany) system was used. The operating conditions are maintained at a voltage of 30.0 kV and a current of 10.0 mA. The increment is 0.02°, the scanning range is 3°～40°, and the scanning speed is 0.1 s/step.

Supersaturated aqueous solutions of BBR, Nnps and Nnps-BBR complexes were prepared. The concentration of BBR in each group was equal, namely 10 mg/mL. After sonicating for 1 h, centrifuge at 16,000 rpm for 10 min, and take the supernatant. The concentration of BBR in the supernatant was determined using the LC-MS method.

The experiment was carried out using the Tianda RC-MD dissolution apparatus (Tianjin, China) based on the rotating basket method, in which the basket speed was maintained at 50 rpm, and the temperature of the dissolution medium was maintained at 37 ± 0.5°C. The sample containing 10 mg BBR (BBR) , Nnps, Nnps-BBR compound) encapsulated in blank capsules and placed in a basket. Then put the basket into a dissolving vessel containing 900 mL of hydrochloric acid buffer (2.0 g sodium chloride, pH 1.2) (simulated gastric juice) or 900 mL of phosphate buffer (0.2 M sodium phosphate, pH 6.8) (simulated intestinal juice). For experiments in hydrochloric acid buffer, collect samples at 5, 10, 15, 20, 30, 45, 60, and 90 minutes. For experiments in phosphate buffer, samples were collected at 2.5, 5, 15, and 30 minutes and 1, 2, 3, 4, and 6 hours, respectively. The sample is filtered through a 0.22-μm filter. After centrifugation (16,000 rpm, 10 minutes), the concentration of BBR in each sample was determined using the LC-MS method.

Cultivate Caco-2 cells until they reach 80-90% confluence. Change the medium 2 hours before the experiment. After washing with warm HBSS, the cells are combined with BBR (10 μg/mL), Nnps (containing 10 μg/mL BBR) or Nnps plus various endocytosis inhibitors (2.5 mm amiloride, a giant pinocytosis) Action inhibitor; 31 100 μg/mL indomethacin, a caveolae-mediated endocytosis inhibitor; 32 10 μg/mL chlorpromazine, a clathrin-mediated endocytosis Inhibitor; 32 5 μM Cytochalasin D, a phagocytosis inhibitor 31). After incubating for 4 h, the medium was removed and the cells were washed with HBSS. After trypsinization, the cells were collected by centrifugation at 1000 rpm for 2 minutes. Then the cells were washed twice with HBSS. Then, the cells were resuspended in cold water and disrupted by repeated freezing and thawing. The concentration of BBR was determined using the LC-MS/MS method, and the protein concentration of each sample was determined using the BCA kit. The concentration of BBR in each group was normalized to the protein content.

Wash an approximately 12 cm long ileum with cold Krebs-Ringer buffer (118 mM NaCl, 25 mM NaHCO3, 1.2 mM MgSO4, 2.5 mM CaCl2, 11 mM glucose, 1.2 mM KH2PO4, and 4.7 mM 6 KCl). , And then ligate at one end. In order to study the transport of BBR from the mucosal side to the serosal side, the inner mucosal side of the intestinal sac was filled with 1 mL of Krebs-Ringer buffer complex containing BBR (10 mg/mL), Nnps or Nnps-BBR, with a corresponding concentration of BBR. To study the transport of BBR from the serosal side to the mucosal side, the intestinal sac was turned over and filled with 1 mL of Krebs-Ringer buffer-BBR complex containing BBR (200 µg/mL BBR) or Nnps or Nnps on the inner serosal side , With the corresponding concentration of BBR. The other end of the ileum was tightly ligated. The capsules were incubated in a Magnus bath containing 20 mL of blank Krebs-Ringer buffer at 37°C. After incubation for 15, 30, 45 or 60 minutes, sample an aliquot of buffer (100 μL) from the Magnus bath. Immediately add an equal volume of blank Krebs-Ringer buffer. The sac length measured after the incubation was used to determine and normalize the concentration of BBR in the obtained sample.

MDCK-MDR1 cells were seeded on Merck Transwell polycarbonate membrane (New Jersey, USA) at a density of 2×105 cells/mL. Culture the cells until tight junctions are formed (transepithelial resistance> 500 Ω·cm2). Before the experiment, replace the medium on both sides of the chamber with warm HBSS solution. Then equilibrate the cells in the incubator for 20 minutes. For the transport test from the apex (AP) to the basolateral (BL) side, add 0.2 mL of HBSS solution containing BBR or Nnps (both with a final concentration of 10 μg/mL BBR) on the AP side, and add 0.7 mL of blank HBSS solution Add to the BL side. For the transport test from the BL to the AP side, add 0.2 mL of blank HBSS solution to the AP side, and add 0.7 mL of HBSS solution containing BBR or Nnps (both with a final BBR concentration of 10 μg/mL) to the BL side. After incubating for 2 or 4 hours, aspirate the solution on both sides and use the LC-MS/MS method to determine the concentration of BBR.

The test materials (BBR and Nnps, the final concentration of BBR are both 10 μg/mL) and intestinal S9 (2 mg/mL) are mixed in 100 μL Tris-HCl (50 mM, pH 7.4) buffer solution, and then pre-incubated in Keep at 37°C for 5 minutes. Then, reduced nicotinamide adenine dinucleotide phosphate (NADPH, 1 mg/mL) was added to start the reaction. After incubation for 0.5 or 1 hour, the incubation was terminated with an equal amount of frozen methanol containing the internal standard carbamazepine. After centrifugation at 16,000 rpm for 6 minutes, the obtained supernatant was diluted to determine the concentration of BBR and desmethylberberine using the LC-MS/MS method. The metabolic stability of BBR is characterized by the concentration of residual BBR and the concentration of desmethylberberine produced.

The mice were randomly divided into three groups, and they were orally orally administered BBR, Nnps-BBR complex, or aqueous solution of Coptis extract. The dose of BBR in each group was 200 mg/kg. The dose of BBR is consistent with the dose in the mouse study, that is, 0.1-0.3 g/kg. 27 Each group of 6 mice was anesthetized with ether at 0.5, 1, 2, 4, 8 or 12 hours after injection. administrative. Collect blood samples from the portal vein or retroorbital venous plexus into a tube containing heparin. The plasma sample was obtained by centrifuging the blood sample at 3000 rpm for 10 minutes at 4°C. Collect mouse liver and homogenize in 10 times volume of water. At the end of the experiment, the mice were euthanized by cervical dislocation. The plasma and liver homogenate samples are stored at -80°C. The LC-MS/MS method was used to determine the concentration of BBR in each sample.

The permeability of BBR in the MDCK-MDR1 experiment is calculated using the following formula: (1)

Where ΔQ is the amount of drug transported during Δt, A is the surface area (0.33 cm2 in this experiment), and C0 is the initial concentration of BBR on the donor side. The unit of Papp is cm/s.

The efflux rate (ER) of BBR in the MDCK-MDR1 experiment is calculated using the following formula: (2)

Non-compartmental analysis was performed using WinNonlin® software (Pharsight, CA, USA) to obtain pharmacokinetic parameters. It should be noted that the pharmacokinetic parameters are calculated based on the average drug concentration at each time point, because mice are not sampled continuously.

The results were expressed as the mean ± standard deviation, and the statistical significance of multiple comparisons with the smallest level of significance (p <0.05) was determined by one-way or two-way analysis of variance (ANOVA).

The residue in the dialysis bag is obtained by dialysis of Coptidis extract. The residue content in Coptis extract was 6.2% (Figure 1A). The residue is mainly composed of protein (>90%). In addition, the content of polysaccharides in the residue is more than 30%. It should be noted that the presence of glucose may lead to an overestimation of protein quantification. 33 This may be one of the reasons why the total content of protein and polysaccharide exceeds 100%. In addition, the residue contains about 8% BBR, which is difficult to remove even after 5 days of dialysis. The freeze-dried powder of the residue is dispersed in 166.6 ± 1.3 nm nanoparticles (ie, Nnps) in water, and the Zeta potential is approximately -12.5 ± 0.2 mV (Figure 1B). Since it is insoluble after denaturation, the purity and molecular weight of the protein forming Nnps cannot be directly determined by gel electrophoresis. From the extract of Rhizoma Coptidis obtained by decoction of herbal tablets at a temperature below 50°C, a purified and soluble protein with a molecular weight of <30 kD was obtained (Figure 1C). After the protein was decocted in boiling water for 1 h, nanoparticles with a size of 176.1 ± 10.1 nm and a Zeta potential of -19.3 ± 0.6 mV were formed, indicating that Nnps is formed by protein denaturation and self-assembly. Under SEM, spherical particles were observed in the Nnps powder, with smaller particles distributed around them (Figure 1D). The mass of the drug formed by small green fluorescent particles was observed under LCFM (Figure 1E), and the dispersed green fluorescent particles were observed in the aqueous solution (Figure 1F). According to reports in the literature, green fluorescence is derived from BBR. 34 In short, the Nnps isolated from the extract of Coptidis Rhizoma are mainly formed by protein denaturation and can adsorb BBR firmly. Figure 1 Characterization of natural nanoparticles (Nnps) isolated from Coptis extract; (A) Content of Nnps in Coptis extract; (B) Size and Zeta potential of Nnps; (C) Protein molecules that form Nnps (CRP, The purity and molecular weight of berberine; (D) scanning electron microscope observation of Nnps powder (10000×); (E) laser confocal fluorescence microscope (6000×) observation of Nnps powder; (F) laser confocal fluorescence microscope (6000×) ×) Observe the aqueous solution of Nnps. In Figure 1D, the large red rectangle shows the enlarged image of Nnps contained in the small red rectangle. In Figures 1E and F, the red dashed rectangles respectively show the enlarged images of Nnps contained in the red dashed small circles.

Figure 1 Characterization of natural nanoparticles (Nnps) isolated from Coptis extract; (A) Content of Nnps in Coptis extract; (B) Size and Zeta potential of Nnps; (C) Protein molecules that form Nnps (CRP, The purity and molecular weight of berberine; (D) scanning electron microscope observation of Nnps powder (10000×); (E) laser confocal fluorescence microscope (6000×) observation of Nnps powder; (F) laser confocal fluorescence microscope (6000×) ×) Observe the aqueous solution of Nnps. In Figure 1D, the large red rectangle shows the enlarged image of Nnps contained in the small red rectangle. In Figures 1E and F, the red dashed rectangles respectively show the enlarged images of Nnps contained in the red dashed small circles.

Under SEM (Figure 2A) and LCFM (Figure 2Ca), long columnar crystals were observed in the BBR powder, but not in the Nnps-BBR composite powder (Figure 2B and Cc). Under LCFM, bamboo leaf-like crystals were observed in the BBR solution (Figure 2Cb), but green fluorescent particles were found in the Nnps-BBR composite solution (Figure 2Cd). DLS analysis shows that the Nnps-BBR complex is dispersed in nanoparticles with a size of 151.3 ± 1.2 nm, and the Zeta potential in water is –11.6 ± 1.2 mV. DSC analysis shows that the melting point of BBR is about 194.97°C, which is consistent with the literature.35 The melting point of the Nnps-BBR composite drops to 175.22°C (Figure 2D). BBR has many characteristic sharp interference peaks in its PXRD pattern, but they are reduced or disappeared in the PXRD pattern of the Nnps-BBR complex (Figure 2E). The above results indicate that Nnps acts as a nanocarrier to adsorb or disperse BBR, thereby promoting its transformation from crystalline to amorphous. Figure 2 Natural nanoparticles (Nnps) changed the crystal form of berberine hydrochloride (BBR). Scanning electron microscope images of BBR (A) and Nnps-BBR (B) (10,000×); (C) BBR powder (a), BBR solution (b), Nnps-BBR powder (c), Nnps-BBR solution (d) Observed by laser confocal fluorescence microscope (6000×); (D) Differential scanning calorimetry images of the physical mixture of BBR, Nnps, Nnps and BBR (Mix), Nnps-BBR complex; (E) BBR, Nnps, Nnps The powder X-ray diffraction image of the physical mixture (mixed) with BBR and the Nnps-BBR composite.

Figure 2 Natural nanoparticles (Nnps) changed the crystal form of berberine hydrochloride (BBR). Scanning electron microscope images of BBR (A) and Nnps-BBR (B) (10,000×); (C) BBR powder (a), BBR solution (b), Nnps-BBR powder (c), Nnps-BBR solution (d) Observed by laser confocal fluorescence microscope (6000×); (D) Differential scanning calorimetry images of the physical mixture of BBR, Nnps, Nnps and BBR (Mix), Nnps-BBR complex; (E) BBR, Nnps, Nnps The powder X-ray diffraction image of the physical mixture (mixed) with BBR and the Nnps-BBR composite.

When the drug is in the amorphous form, it generally has better solubility and dissolution than the crystalline form. 36 As shown in Figure 3A, the solubility of BBR in the Nnps-BBR complex is significantly higher than that of pure BBR. The dissolution rate of BBR in artificial gastric juice (Figure 3B) and intestinal juice (Figure 3C) was only 7.5% and 1.8% at 15 minutes, and 35.3% and 60.9% at the end point (1.5% or 6 h). However, The dissolution rate of BBR in Nnps increased to 50.8% and 31.5% at 15 minutes, and to 69.1% and 85.4% at the end point, respectively. In addition, the dissolution rate of BBR in the Nnps-BBR complex increased to 54.55% and 22.55% at 15 minutes, and to 60.0% and 81.37% at the end of the dissolution, respectively. Figure 3 The effect of natural nanoparticles (Nnps) on the solubility (A) and dissolution curve of berberine hydrochloride (BBR) in simulated gastric juice (B) or intestinal juice (C) (mean ± SD, n = 6). ** p <0.01 and BBR.

Figure 3 The effect of natural nanoparticles (Nnps) on the solubility (A) and dissolution curve of berberine hydrochloride (BBR) in simulated gastric juice (B) or intestinal juice (C) (mean ± SD, n = 6). ** p <0.01 and BBR.

Nanoparticles are absorbed mainly through endocytosis. 37 Indomethacin significantly reduced the absorption of BBR by Caco-2 cells (Figure 4A, p <0.01), indicating that the BBR carried by Nnps is mainly absorbed through cavern-mediated endocytosis. In vitro absorption experiments based on mouse intestinal sacs found that the intestinal absorption of BBR in the Nnps and Nnps-BBR complex was significantly better than pure BBR (Figure 4B, p <0.01), indicating that Nnps can adsorb BBR and act as a nanocarrier to promote BBR Absorption. In addition, Nnps significantly reduced BBR outflow from the intestinal sac (Figure 4C, p <0.01). Verapamil is a typical P-gp inhibitor,38 significantly reducing the efflux of BBR in the BBR and Nnps groups (Figure 4C). However, the effect on the Nnps group was less than that on the BBR group, indicating that Nnps can reduce P-gp-mediated BBR efflux (Figure 4C). The results of in vitro experiments based on MDCK-MDR1 cells (Table 1) show that the Papp of BBR is lower than 10-6 cm/s, indicating that its permeability is poor. The ER of BBR was as high as 25.8 at 2 hours and 39.5 at 4 hours. According to the recommendations of the International Transporter Alliance, if ER ≥ 2.39, the compound is considered to be a potential P-gp substrate. The ER of BBR is much greater than the critical value 2, indicating that P-gp-mediated efflux significantly limits the absorption of BBR . In contrast, Nnps significantly reduced Papp and ER from BL to AP (all p <0.01), confirming that Nnps inhibited P-gp-mediated BBR efflux. The results of in vitro metabolism experiments showed that compared with the BBR group, the metabolic rate of BBR in Nnps (Figure 4D) and the production of desmethylberberine (Figure 4F) were significantly reduced, and the metabolic rate of BBR was reduced by 29.27%. The production of methyl berberine decreased by 22.39%. The elimination half-life of BBR was significantly extended from 149 minutes to 216 minutes (Figure 4E). In summary, these results indicate that Nnps changed the absorption form of BBR, reduced its intestinal efflux, and improved its intestinal metabolic stability. Table 1 The effect of natural nanoparticles (Nnps) on the apparent permeability (Papp) and efflux rate (ER) of berberine hydrochloride (BBR) (mean ± SD, n = 3) Figure 4 Natural nanoparticles (Nnps) Intestinal effects on the absorption of berberine hydrochloride (BBR) (mean ± standard deviation); (A) the effect of endocytosis inhibitors on the uptake of BBR (10 μg/mL) in Caco-2 cells (n = 3); (B) Absorption of BBR (10 mg/mL) in the intestinal sac (n = 4); (C) In the presence or absence of 100 μg/mL verapamil (n = 4), BBR (200 μg/mL) outflow in the intestinal sac; (D) elimination of BBR (10 μg/mL) in intestinal S9 (n = 3); (E) BBR (10 μg/mL) in intestinal S9 (n = 3) Elimination half-life in (F) Demethylberberine production after incubation of BBR (10 μg/mL) with intestinal S9 (n = 3). AMI, amiloride (2.5 mM); BBR, berberine hydrochloride; CHL, chlorpromazine (10 μg/mL); CYT, cytochalasin D (5 μM); IND, indomethacin (100 μg) /mL); Nnps, natural nanoparticles. A, * or **p <0.01 and Nnps. E, *p <0.05 and BBR.

Table 1 The effect of natural nanoparticles (Nnps) on the apparent permeability (Papp) and efflux rate (ER) of berberine hydrochloride (BBR) (mean ± standard deviation, n = 3)

Figure 4 The effect of natural nanoparticles (Nnps) on the intestinal absorption of berberine hydrochloride (BBR) (mean ± standard deviation); (A) endocytosis inhibitors on Caco-2 cells (n = 3) in BBR (10 μg/mL) the effect of ingestion; (B) BBR (10 mg/mL) absorption in the intestinal sac (n = 4); (C) in the presence or absence of 100 μg/mL verapamil (n = 4) ), the outflow of BBR (200 μg/mL) in the intestinal sac; (D) elimination of BBR (10 μg/mL) in intestinal S9 (n = 3); (E) BBR (10 μg/mL ) Elimination half-life in intestinal S9 (n = 3); (F) Demethylberberine production after incubation of BBR (10 μg/mL) with intestinal S9 (n = 3). AMI, amiloride (2.5 mM); BBR, berberine hydrochloride; CHL, chlorpromazine (10 μg/mL); CYT, cytochalasin D (5 μM); IND, indomethacin (100 μg) /mL); Nnps, natural nanoparticles. A, * or **p <0.01 and Nnps. E, *p <0.05 and BBR.

Nnps had no significant effect on the pharmacokinetics of oral BBR in the systemic circulation (Figure 5A, Table 2). However, compared with the BBR treatment group, the peak concentration of BBR (Cmax, 1182.3 vs 310.2 ng/mL) and the exposure level (AUC0–12 h, 2842.8 vs 1447.0 ng·h/mL) were significantly higher in the Nnps-BBR treatment group. The portal vein (Figure 5B, Table 2) shows that Nnps promotes the intestinal absorption of BBR. In addition, Nnps caused higher exposure levels of BBR in the liver of mice (95,443.2 and 43,586.2 ng·h/g liver) (Figure 5C, Table 2). In addition, the pharmacokinetic parameters of the Nnps-BBR group (Table 2), including Cmax and AUC0-12 h, were similar to or slightly better than the Coptidis treatment group. In short, Nnps improved the pharmacokinetics of oral BBR in mice. Table 2 The pharmacokinetic parameters of berberine hydrochloride (BBR) in the mouse systemic circulation, portal vein and liver of 200 mg/kg oral BBR, nanoparticle and BBR complex (Nnps-BBR), and Coptis extract, including the corresponding dose of BBR (Mean ± standard deviation, n = 6) Figure 5 Receiving 200 mg/kg oral BBR, a complex of natural nanoparticles (Nnps) and BBR (Nnps-BBR) and Coptis extract, all containing the corresponding dose of BBR (average Value ± standard deviation, n = 6).

Table 2 The pharmacokinetic parameters of berberine hydrochloride (BBR) in mice systemic circulation, portal vein and liver of 200 mg/kg oral BBR, nanoparticle and BBR complex (Nnps-BBR), Coptidis extract containing the corresponding dose of BBR (Mean ± standard deviation, n = 6)

Figure 5 The concentration-time curve of berberine hydrochloride (BBR) in the systemic circulation (A), portal vein (B) and liver (C) of mice receiving 200 mg/kg oral BBR (Nnps complex) BBR (Nnps-BBR) and Coptis extract, they both contain the corresponding dose of BBR (mean ± standard deviation, n = 6).

In this study, the Nnps isolated from the extract of Coptidis Rhizoma were mainly formed by the denaturation of proteins (probably glycosylated proteins) with a molecular weight slightly less than 30 kDa. Since the protein is very pure, Nnps of relatively uniform size can be quickly obtained by simple methods (including centrifugation, filtration, and dialysis) instead of further purification using size exclusion high performance liquid chromatography (SEC-HPLC). The size of 18 Nnps is less than 200 nm, which helps prevent them from being engulfed by reticuloendothelial phagocytes (macrophages). 40 The Zeta potential of these nanoparticles indicates their insufficient stability, leading to the aggregation of nanoparticles observed in Figure 2Cd. However, considering that Nnps will be taken immediately after preparation, a large accumulation of Nnps can be avoided. SEM observations, DSC and PXRD experiments show that Nnps promotes the transformation of BBR to an amorphous form. Therefore, in Coptis extract, Nnps can be used as a crystallization inhibitor of BBR. In other words, Coptis extract can be regarded as a solid dispersion of BBR with Nnps as the carrier. In addition, Nnps can form complexes with BBR. According to the literature, 41 Nnps and BBR can form a hydrophobic interaction. The dispersion of Nnps-BBR complex into nanoparticles in water once again shows that Nnps has had a major and fundamental impact on the existing form of BBR.

About 56% of BBR is directly excreted from the body after oral administration, which is related to the poor solubility of BBR. 27 The usual dose of BBR in mice is 200 mg/kg. 27 Therefore, if BBR is 0.2 mL/10 g body weight, its concentration should be 10 mg/ml. However, the solubility of BBR in this study is only about 3.7 mg/mL, which means that more than half of the BBR is undissolved. By forming a solid dispersion with BBR, Nnps significantly improves the solubility and solubility of BBR.

Nanoparticles are absorbed by intestinal cells mainly through phagocytosis, macropinocytosis, pit-mediated endocytosis, and clathrin-mediated endocytosis 37. They are absorbed by cytochalasin D and 31 amilor, respectively. Li, 31 indomethacin, 32 and chlorpromazine 32 inhibited. Based on experiments using Caco-2 cells, it was found that Nnps carries BBR and promotes its absorption through the indomethacin-sensitive endocytosis mechanism. In other words, Nnps changed the form of BBR absorption in the intestine, from passive diffusion26 to endocytosis. Generally, caveolae mediates the uptake of nanoparticles smaller than 100 nm in size. 42 However, some studies have shown that caveolae mediates the endocytosis of nanoparticles larger than 200 nm43 or even 500 nm. 44 Importantly, caveolae-mediated endocytosis can bypass lysosomal degradation, 37 indicating that Nnps can be transported into the circulation in the form of nanoparticles after being absorbed by intestinal epithelial cells. In this study, chlorpromazine and cytochalasin D promoted the absorption of BBR by Nnps, which may be related to its inhibitory effect on the efflux transporter P-gp. Given that chlorpromazine and cytochalasin D are P-gp substrates, 45, 46 they can competitively inhibit the efflux of BBR.

According to reports in the literature, 43.5% of oral BBR is cleared in the intestine. 27 Cytochrome P450 enzymes (CYPs), especially CYP3A, play a major role in the phase I metabolism of BBR. 47 There are a variety of CYPs in intestinal epithelial cells, especially CYP3A. 48 Our research results show that Nnps significantly inhibits the phase I metabolism of BBR in the intestine, which is beneficial to increase its intestinal absorption. This result is consistent with previous reports that curcumin loaded in polymer nanoparticles is not metabolized. 49 In addition, there are many efflux transporters in intestinal epithelial cells, such as P-gp, which significantly reduces the absorption of their substrates. In addition, there is a synergistic effect between efflux transporters and metabolic enzymes, which together mediate the intestinal elimination of certain drugs. 50 For example, the phase I metabolic enzymes CYP3A4 and P-gp have many overlapping substrates. P-gp can affect drug metabolism in the intestine by reducing transcellular transport and increasing the contact between the substrate and the intestinal cell tip CYP3A451. Unfortunately, BBR is a substrate of CYP3A4 and P-gp; therefore, it is eliminated by the combined action of the two during intestinal absorption. 27 In this study, Nnps significantly reduced the outflow of BBR from the mouse intestinal sac. In addition, Nnps significantly reduced the Papp of BBR from the basal side to the luminal side, and significantly reduced the ER of BBR in MDCK-MDR1 cells. Since MDCK-MDR1 cells only express P-gp, this result confirmed that Nnps significantly inhibited P-gp-mediated BBR efflux. Similarly, after loading nanoparticles separated from green tea infusion, doxorubicin can bypass the efflux function of P-gp. 18,52

Pharmacokinetic studies in mice have shown that Nnps significantly promotes the intestinal absorption of BBR, which is manifested as a significant increase in portal BBR exposure levels (AUC0-t and Cmax). Thereafter, due to the increase in intestinal absorption, the distribution of BBR in the liver increases. However, the increase in BBR in the systemic circulation is not as significant as in the portal vein and liver. This result may be related to the active uptake of BBR by tissues including liver tissue, resulting in a poor dose-exposure relationship of BBR in the systemic circulation. 28 The pharmacological effects of BBR in reducing blood sugar and blood lipids are the focus of research. Current research. 53 The liver is the main tissue involved in glucose and lipid metabolism. 54 Therefore, the increase in BBR exposure in the liver indicates that Nnps can enhance the pharmacological effects of BBR in reducing blood sugar and blood lipids. In addition, the exposure levels of the portal vein and liver of the mice in the Nnps-BBR treatment group were comparable to those in the Coptis extract treatment group, indicating that Nnps has a strong ability to improve the pharmacokinetic properties of BBR.

The interaction between the components of herbal extracts has always been a concern, and is often used to explain the synergistic effects of traditional Chinese medicine or the mechanism of reducing toxicity. 55 These interactions may occur at the pharmacodynamic, pharmacokinetic, and chemical level. At the level of pharmacodynamics, a multi-component and multi-target point of view is proposed. 56 At the pharmacokinetic level, it is usually reported based on the interaction between drug-metabolizing enzymes and transporter components. 57 At the chemical level, it is reported that some ingredients, such as BBR and glycyrrhizic acid, 58 can form complexes based on intermolecular interactions. In addition, BBR can form nanoparticles with baicalin and wogonin, which has a significant impact on its antibacterial effect. 59 However, the above-mentioned research is limited to the study of interactions between small molecular components in herbal extracts. This study found that a protein in Coptis extract can interact with BBR, and for the first time systematically studied the interaction mechanism from the two levels of chemistry and pharmacokinetics. Given that Nnps formed by macromolecules are widely present in herbal extracts, we believe that this research will stimulate more related research and help reveal the mechanism of interaction between herbs.

To sum up, the Nnps in the extract of Coptis Rhizoma Coptidis changes the existing form of BBR and promotes its transformation from crystal to amorphous form, thereby improving its solubility and solubility. Nnps also carries BBR and promotes its uptake by intestinal epithelial cells through caveola-mediated endocytosis, and significantly reduces the intestinal metabolism of BBR and P-gp-mediated efflux. These mechanisms act synergistically to ultimately promote the intestinal absorption of BBR and increase its exposure level in the body. This research helps to reveal the mechanism of interaction between herbs in Coptis extract.

BBR, berberine hydrochloride; BCA, quinolinic acid; Caco-2, human colon adenocarcinoma cells; CYPs, cytochrome P450 enzymes; DMEM, Dulbecco's modified Eagle medium; DMSO, dimethyl sulfoxide; DSC, poor Scanning calorimetry; ER, efflux rate; ESI, electrospray ionization; HBSS, Hank's balanced salt solution; LCFM, laser confocal fluorescence microscope; LC-MS, liquid chromatography-mass spectrometry; LC-MS/MS , Liquid chromatography tandem mass spectrometry; MDCK-MDR1, Madin-Darby canine kidney cells stably expressing the transporter P-glycoprotein; NADPH, reduced nicotinamide adenine dinucleotide phosphate; Nnps, natural nanoparticles; Papp, permeation Sex; P-gp, p-glycoprotein; PXRD, powder X-ray diffraction; SEM, scanning electron microscope; Chinese medicine, Chinese medicine.

The data set used and/or analyzed in the current research can be obtained from the corresponding author Bing-Liang Ma upon reasonable request.

All animal experiment protocols were approved by the Institutional Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine (PZSHUTCM19011105). All experiments were carried out in accordance with the guidelines for the care and use of laboratory animals in Shanghai University of Traditional Chinese Medicine.

This work was funded by the Shanghai Natural Science Foundation (17ZR1430400).

Zhao Jing and Zhao Qing share the first author. The author did not declare a conflict of interest.

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