Introduction
Shaoyao Gancao Decoction (SGD) is a famous traditional Chinese medicine (TCM) formula originating from the Treatise on Febrile diseases written by Zhongjing Zhang during the Han Dynasty.1,2 It is composed of Paeoniae Radix Alba (PRA, called Baishao in China) and Glycyrrhizae Radix et Rhizoma (GRR, called Gancao in China) at a ratio of 1:1, and is widely used in the treatment of digestive system diseases and gynecological disorders.2,3 Recent studies have demonstrated that SGD possesses multiple pharmacological effects, including spasmolysis, analgesia, and anti-inflammatory effects, and has various chemical components, including paeoniflorin (Pae), oxypaeoniflorin, albiflorin, benzoylpaeoniflorin, glycyrrhizic acid (GL), liquiritin apioside, liquiritin, liquiritigenin.1–4 However, these components are predominantly glycosides or flavonoids, which exhibit poor oral bioavailability because of their high polarity or low solubility. In particular, Pae, a P-glycoprotein (P-gp) substrate, was demonstrated to be extremely poor absorption with absolute oral bioavailability ranging from 3% to 4%.5,6
Notably, TCM herbal formulas exhibit an intriguing characteristic that certain phytochemicals with inherently low bioavailability show dramatically increased absorption when orally administered with TCM formula decoctions. Representatively, the bioavailability of Pae was significantly improved by the oral administration of SGD,7 but the mechanism underlying the bioavailability improvement remains unclear. There have been some studies on the potential mechanisms by which active ingredients of licorice affect Pae absorption. However, there are contradictory findings regarding the effects of licorice constituents on P-gp. Glycyrrhetinic acid (GA) and 18β-GA (a predominant conformation of GA) have shown P-gp inhibitory activity in certain studies.8,9 In contrast, other studies have reported that GA activated the function and expression of P-gp.10,11 Additionally, GL has been reported to decrease the absorption of Pae by inducing the activity of P-gp.12 In another study, GL exhibited concentration-dependent effects on intestinal Pae absorption: 1 mM GL significantly increased absorption in the jejunum after 45 min and in the ileum after 90 min, whereas 100 μM GL inhibited absorption in the jejunum.13 Therefore, the underlying mechanisms by which oral administration of SGD improves bioavailability of Pae remain to be elucidated due to these inconsistent findings.
Recently, self-assemblies of TCM, also known as supramolecules and natural nanoparticles, have received growing attention as they can not only help elaborate the scientific connotation of TCM compatibility, but also be used as drug carriers to improve the bioavailability and biological activities of medicinal components.14–17 Self-assemblies in TCM decoctions, particularly in TCM formulas, result from high-temperature boiling-induced non-covalent interactions (hydrogen bonding, electrostatic forces, ionic interactions, and hydrophobic effects) among the active components.15,16 Our previous study has demonstrated that GL can self-assemble into nanomicelles, significantly enhancing the intestinal absorption and bioavailability of Pae.18 Glycyrrhiza protein (GP) has also been reported to self-assemble into functional nanoparticles that are capable of significantly increasing solubility, promoting absorption, and enhancing the bioactivity of drugs.19–22 The self-assembled GP nanoparticles were also found to increase the intestinal absorption of Pae.23 However, the formation of self-assembled nanoparticles in SGD (SGD-SAN) is not achieved solely by GL or GP, but rather results from the synergistic interaction of multiple components. It remains unclear whether the nano-assemblies in SGD differ fundamentally from either GL self-assembled nanomicelles (GL-SNM) or GP self-assembled nanoparticles (GP-SAN).
Therefore, the present study was performed to systematically compare the in vitro and in vivo characteristics of three nano-assemblies of different components from SGD (SGD-SAN, GL-SNM, and GP-SAN), with particular emphasis on their differential effects on oral absorption of Pae. SGD-SAN was separated and then identified for its composition. GL-SNM and GP-SAN were self-assembled according to the main components obtained from the composition analysis of SGD-SAN and loaded with Pae, named Pae GL-SNM and Pae GP-SAN, respectively. The SGD-SAN, Pae GL-SNM, and Pae GP-SAN were characterized in terms of particle size, zeta potential, morphology, drug loading, and in vitro release behavior. The single-pass intestinal perfusion and pharmacokinetic studies of SGD-SAN, Pae GL-SNM, and Pae GP-SAN following oral administration were performed to evaluate their absorption-enhancing effect. These comprehensive comparisons will elucidate the structure-function relationships underlying SGD’s enhancement of Pae absorption, which is critical for revealing the synergistic mechanisms of multi-component interactions in TCM and clarifying the scientific basis of TCM compatibility. Moreover, clarifying these nano-assemblies’ contributions may guide the development of novel TCM-based drug delivery systems and more effective TCM-derived formulations, providing support for the modern application of TCM.
Materials and Methods
Materials
PRA and GRR were purchased from Jiangxi Peng’s Gaoyaotang Sliced Herb Co., Ltd. The herbal materials were authenticated by the Chief Pharmacist Ping Ying from the Department of Pharmacy, Jiangxi Provincial People’s Hospital (the First Affiliated Hospital of Nanchang Medical College), PR China. Pae (purity > 95%), GL (purity > 95%), Pae reference standard (purity > 98%), and puerarin (internal standard, purity > 98%) were obtained from Baoji Herbest Bio-Tech Co., Ltd (Xian, PR China). HPLC-grade methanol and acetonitrile were purchased from Sigma-Aldrich (USA). 3500 Da dialysis bag was obtained from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). All other chemicals used were of analytical grade.
Animals
Male Sprague-Dawley rats (weight 180–200 g, 6 weeks old) were obtained from SPF (Beijing) Biotechnology Co., Ltd. (Beijing, China). All rats were housed in an environmentally controlled breeding room (25 ± 2 °C, 60% humidity, and 12 h cycle of light and dark) and received ad libitum access to water and food. All animal experiments were conducted in compliance with the NIH Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Ethics Committee of Nanchang Medical College (No. NYLLSC20240408).
Separation and Composition Analysis of SGD-SAN
Separation of SGD-SAN
SGD-SAN was separated as follows:24 PRA and GRR herbs were weighed at a ratio of 1:1 (25 g + 25 g) and soaked in deionized water (500 mL) for 30 min. The mixture was boiled twice with 10- and 8-fold volumes (v/w) of deionized water (500 mL and 400 mL per 50 g herbs), each for 1 h. After filtration through eight layers of gauze, the two decoction liquids were combined and concentrated to 1 g/mL (crude drug equivalent) by rotary evaporation at 50 °C under reduced pressure (−0.08 MPa). After SGD preparation, SGD-SAN was obtained using a combined centrifugation-dialysis approach. SGD (50 mL) was centrifuged at 4000 rpm for 30 min to remove the precipitates. The supernatant (5 mL) was dialyzed against 200 mL of deionized water for 30 min using a 3500 Da molecular weight cutoff (MWCO) dialysis bag (25 mm flat width) at room temperature with constant agitation at 200 rpm, and then the liquids in dialysis bag was centrifuged at 13000 rpm for 30 min to remove the precipitates. The dialysis and centrifugation operations were repeated twice. Through the above operations, the liquid inside the dialysis bag was freeze-dried and collected as SGD-SAN.
Composition Analysis of SGD-SAN
The composition of SGD-SAN was analyzed using a BCA assay kit, phenol-sulfuric acid method, and high-performance liquid chromatography (HPLC). The protein content of SGD-SAN was determined using a BCA assay kit according to the manufacturer’s instructions. The polysaccharide content of the SGD-SAN was measured by the phenol-sulfuric acid method.25 About of 20 mg SGD-SAN was dispersed in 10 mL of methanol, followed by ultrasonication for 10 min. After centrifugation at 8000 rpm for 10 min, the supernatant was filtered through 50 nm filters (Sigma-Aldrich, St Louis, MO, USA) for quantitative analysis of the main components by HPLC using an LC-20AD HPLC system (Shimadzu, Tokyo, Japan). Chromatographic separation was performed on an Inertsil® ODS-3 column (250 mm × 4.6 mm, 5 µm, Shimadzu, Tokyo, Japan) using a mobile phase of acetonitrile and 0.1% phosphoric acid solution with gradient elution (Table 1). The flow rate was maintained at 1.0 mL/min, with the column temperature set at 30 °C. Detection was performed at a wavelength of 237 nm and the injection volume was 10 μL.
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Table 1 Gradient Elution Conditions for Mobile Phase
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Extraction of GP and Preparation of Pae GP-SAN
GP extraction from GRP was performed by the acetone precipitation method22 with some modifications. Briefly, GRP decoction pieces were mixed with deionized water at ratio of 1:5 (w/v) and extracted under ultrasonication for 30 min at 60 °C. The resulting mixture was filtered through a double-layer filter cloth and centrifuged at 4,000 rpm for 15 min to remove residues. The supernatant was precipitated with 1.5 times volume of acetone, incubated at −20 °C for 30 min. The precipitates were collected by centrifugation at 4,000 rpm for 15 min and then freeze-dried to yield GP. The obtained GP was determined by sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were mixed with loading buffer and then heated at 95 °C for 10 min for protein denaturation. The denatured protein samples were separated by SDS-PAGE at 80 V to 120 V, and the protein bands were stained with Coomassie Brilliant Blue and compared with the protein standard.
The Pae GP-SAN was prepared by an ultrasonic dispersion method. Briefly, 16 mg of GP and 12 mg of Pae were added to 4 mL of deionized water and sonicated (JK-50B, Hefei Jinnick Medical Technology Co., Ltd., Hefei, China) at 250 W (50 Hz) for 25 min. After cooling to room temperature, the mixture was filtered through a 0.80 µm filter to obtain Pae GP-SAN.
Preparation of Pae GL-SNM
The Pae GL-SNM was fabricated via the film dispersion method, with a formulation consistent with that of our previous study.18 Briefly, GL (40 mg) was dissolved in 10 mL of absolute ethanol in a 50 mL pear-shaped flask and then evaporated by rotary evaporator (RE-2000B, Shanghai Yarong Biochemistry Instrument Factory, Shanghai, China) at 50 °C to obtain a thin film of GL. The obtained film was hydrated with 9.2 mL of deionized water through ultrasonication in a 50 °C water bath to form a micellar suspension. Then, 10 mg of Pae was dissolved in 0.8 mL of ethanol and added dropwise to the micellar suspension at 50 °C under continuous magnetic stirring (MS-H280-Pro, Dragon Laboratory Instruments Ltd., Beijing, China) at 500 rpm. After cooling to room temperature, the resulting mixture was sonicated for 30 min in an ice-water bath using an ultrasonic cell disintegrator (JY92-IIN, Ningbo Scientz Biotechnology Co., Ltd., China) at 240 W with a 3 s pulse interval. The ethanol was removed by rotary evaporation, and the volume was adjusted to 10 mL with deionized water. Finally, Pae GL-SNM was obtained by filtration through 0.80 µm microporous membrane filters.
Characterization of Nano-Assemblies
Size Distribution and Zeta Potential Analysis
The particle size and polydispersity index (PDI) of SGD-SAN, Pae GP-SAN, and Pae GL-SNM were determined by dynamic light scattering (DLS) using a Zeta-sizer (Malvern Zetasizer Pro, Worcestershire, UK). Samples were appropriately diluted with deionized water, equilibrated at 25 °C for 2 min prior to measurement, and analyzed at 25 °C with a scattering angle of 173°. Zeta potential was estimated by determining the electrophoretic velocity of the particles using the same instrument.
Morphology Evaluation
The morphologies of SGD-SAN, Pae GP-SAN, and Pae GL-SNM were observed by transmission electron microscopy (TEM, Tecnai G2F20, FEI, Eindhoven, Netherlands). After appropriate dilution with deionized water, one drop of the sample was deposited onto a 200-mesh copper grid, blotted with filter paper, and dried at room temperature. Images were obtained by TEM at 80 kV for morphological evaluation.
Encapsulation Efficiency and Drug Loading
The drug encapsulation efficiency (EE) and drug loading (DL) of Pae GP-SAN and Pae GL-SNM were determined by ultrafiltration method.18,26 Briefly, 0.4 mL of sample was placed into the upper chamber of ultrafiltration centrifugal tube tubes (3 kDa, Millipore Corporation, Billerica, MA, USA) and centrifuged at 8000 rpm for 10 min. The filtrate was diluted 10-fold with methanol and the free Pae concentration was quantified by HPLC (LC-20AD, Shimadzu, Tokyo, Japan). The chromatographic separation was carried out on Inertsil® ODS-3 column (250 mm × 4.6 mm, 5 µm, Shimadzu, Tokyo, Japan) with column temperature maintained at 40 °C and mobile phase of acetonitrile and 0.1% phosphoric acid (17: 83, v/v) at a flow rate of 1 mL/min. Detection was performed at 231 nm with an automated injection volume of 20 μL. The total Pae content in Pae GL-SNM or Pae GP-SAN was measured after extraction with 50-fold methanol in an ultrasonic bath. The EE and DL were calculated as follows:
where Wt is the total amount of Pae in Pae GP-SAN or Pae GL-SNM, Wf is the amount of free Pae unencapsulated in Pae GP-SAN or Pae GL-SNM, Wc is the total amount of GL or GP added. The drug loading (DL) of SGD-SAN was calculated by the Pae content in SGD-SAN, whereas the drug encapsulation efficiency (EE) was calculated as the ratio of the Pae content in SGD-SAN to the total Pae in SGD.
In vitro Drug Release
The Pae release from SGD-SAN, Pae GP-SAN, and Pae GL-SNM in vitro was investigated using a dialysis method and compared with that of Pae solution. The simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 6.8) were selected as release media, respectively. All samples with dose equivalent to 10 mg Pae were placed into a dialysis bag (MWCO: 3500 Da; flat width: 25 mm, effective length: 30 mm). The bag was mounted with its symmetrical axis perpendicular to the direction of horizontal stirring to minimize boundary layer effects. Both ends were sealed with clips and immersed into 200 mL of release medium maintained at 37 ± 1 °C, and stirred at 100 rpm. At various time points (0.25, 0.50, 0.75, 1, 2, 4, 6, 8, and 12 h), 1 mL aliquots were withdrawn and replaced with an equal volume of media. Each sample was filtered through a 50 nm filter, and the Pae concentration was determined by the same HPLC method as described in the determination of EE and DL. Drug release profiles were plotted as the cumulative % drug release versus time.
Single-Pass Intestinal Perfusion Study
The intestinal absorption of SGD-SAN, Pae GP-SAN, and Pae GL-SNM was investigated by in situ single-pass intestinal perfusion method18,27,28 and compared with that of Pae solution. Male SD rats were fasted for 12h with free access to water before the perfusion experiment, and then were anesthetized by intraperitoneal injection of pentobarbital sodium (40 mg/kg). The anesthetized rats were fixed in a supine position on a homeothermic blanket to maintain normal body temperature, and a midline incision of the abdomen was made. Approximately 10 cm segment of either the jejunum or ileum was carefully cannulated on two ends with plastic tubing and ligated using silk sutures. The dissected intestinal segments were gently rinsed with 37 °C saline to clear the segment and then attached to a peristaltic pump (L100-1S-1/DG-2, Baoding Longer Precision Pump Co., Ltd., Baoding, China). The entire surgical area was covered with a piece of sterile absorbent gauze wetted with saline (37 ± 1°C). Initially, the intestinal segment was perfused with blank Krebs-Rings (K-R) solution at a flow rate of 0.2 mL/min for 30 min to remove any residue. Then, the K-R solution containing different Pae samples (SGD-SAN, Pae GL-SNM, Pae GP-SAN, and Pae solution) at the same Pae concentration of 20 µg/mL29 were perfused at a flow rate of 0.2 mL/min for 30 min to ensure steady-state conditions, and the outlet perfused samples were collected in pre-weighed vials at 15 min intervals up to 90 min and then weighed. The perfusion rate of 0.2 mL/min is designed to simulate the physiological state, reduce intestinal injury, and is physiologically equivalent to the 2.0–3.0 mL/min rate used in human jejunal perfusion.30–32 The collected samples were stored at −20 °C until analysis by the same HPLC method as described in determination of EE and DL. Finally, the animals were euthanized with an overdose of isoflurane, and the length and inner diameter of the intestinal segment were measured. The gravimetric method was used to determine net water flux (water absorption and efflux in the intestinal segment).27–29 The absorption rate constant (Ka) and effective permeability coefficient (Peff) of Pae in intestinal segments were calculated using the following equations:
where 
and 
are the concentrations of the test drug in the effluent perfusate through the inlet and outlet tubes (μg/mL), respectively. 
and 
represent the inlet and outlet volumes of the effluent perfusate (mL), respectively. 
is the perfusion volume flow rate (mL/h). 
and 
are the radius and the length of the perfused intestinal segment, respectively.
Pharmacokinetics Study
All rats were acclimated for one week and fasted for 12 h before the experiments. Twenty-four rats were randomly divided into four groups of six animals per group and received Pae solution, SGD-SAN, Pae GL-SNM, and Pae GP-SAN via gavage at a dose of 100 mg/kg Pae.18 After administration, approximately 0.5 mL of whole blood was collected from the orbital venous plexus at 0.083, 0.167, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 12 and 24 h, and then placed into heparinized tubes. The plasma samples were obtained by centrifugation at 5000 rpm for 10 min, and stored at −80 °C until analysis.
The plasma samples were processed according to our previous study,18 and the Pae concentrations in plasma was determined by a validated HPLC/MS/MS system with puerarin as an internal standard (IS). An LC-30AD HPLC system (Shimadzu, Tokyo, Japan) was used for chromatographic separation with a Shim-pack Scepter C18 column (2.1 × 100 mm, 3 µm particle size, Shimadzu, Tokyo, Japan) maintained at 40 °C. The mobile phases consisted of 0.1% aqueous formic acid (A) and methanol (B) with gradient washing as follows: 0 ~ 1.1 min, 5%B; 1.1 ~ 2.4 min, 5 ~ 95% B; 2.4 ~ 4.0 min, 95%B; 4.0 ~ 4.5 min, 95 ~ 5%B; 4.5 ~ 6.0 min, 5%B. The flow rate was 0.3 mL/min and the injection volume was 5 µL. A TRIPLE QUADTM 4500MD mass spectrometer (AB SCIEX, Massachusetts, USA) was interfaced via electrospray ionization (ESI) source operating in positive ion mode at 550°C. The nebulizer gas (Gas 1), heater gas (Gas 2), curtain gas (CUR), and collision activated dissociation gas (CAD) were set to 55, 55, 33, and 8 instrument units, respectively. The quantification of Pae and Pue (IS) were quantified in the MRM mode using the pseudo-molecular (Q1) to fragment (Q3) ion transitions and the optimal declustering potential (DP), collision energy (CE), collision cell exit potential (CXP), and entrance potential (EP), as shown in the following conditions (Table 2).
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Table 2 Mass Spectrometry Conditions
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Determination of Dominant Driving Forces in Self-Assemblies
To elucidate the dominant driving forces for the formation of SGD-SAN, Pae GP-SAN, and Pae GL-SNM, chemical interference agents (NaCl, urea, and Tween 20) were added, coupled with particle size detection, to identify the types of intermolecular forces in the self-assemblies.20,33,34
Statistical Analysis
The main pharmacokinetic parameters were calculated by DAS 2.0. All the experimental data were expressed as the mean ± standard deviation (mean ± SD). The SPSS 21.0 software was used to analyze the statistical data. First, the normality test and homogeneity of variance test of the data were performed. If the data meet the assumptions of normal distribution and homogeneity of variance, one-way analysis of variance (ANOVA) followed by the least significant difference post-hoc test was used to compare the differences among these groups. Otherwise, it was determined by Games–Howell test (heterogeneous variance) or Mann–Whitney U-test (non-normal distribution). Probability values P < 0.05 meant the difference was statistically significant.
Results and Discussions
Separation and Composition Analysis of SGD-SAN
According to our separation method, the average yield of SGD-SAN is (12.93 ± 0.58) %. Nine active ingredients were determined by HPLC. As shown in Figure 1A, the contents of isoliquiritin, liquiritigenin, benzoylpaeoniforin, and isoliquiritigenin were less than 0.2%, the contents of alibiflorin, liquiritin apioside, and liquiritin were about 1.25%, 0.97%, and 1.18%, while the contents of Pae and GL were 4.54% and 4.32%, respectively. These results suggested that Pae and GL were the main active ingredients. The protein content of SGD-SAN determined by BCA kit was about 61.91% (Figure 1B), accounting for most of the weight of SGD-SAN. In addition, the polysaccharide content of SGD-SAN was about 37.58%, while total content of 9 kinds of active ingredients was 12.69% (Figure 1B). The total content of all ingredients exceeded 100% (112.18%), which may be attributed to the overestimation of protein quantification owing to the presence of glucose35 and repeated measurement of part carbohydrate components due to the presence of glycosides.
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Figure 1 Composition analysis of SGD-SAN. (A). Composition of SGD-SAN with standard substances by HPLC (Data are presented mean ± SD.). (B). Proportion of proteins, polysaccharide, and active ingredients in SGD-SAN (Data are presented mean ± SD.). (C). SDS-PAGE bands of GP.
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Preparation of Pae GP-SAN and Pae GL-SNM
To clarify the effects of nano-assemblies in SGD on Pae bioavailability, two new nanoformulations were prepared based on the chemical composition of SGD-SAN and their self-assembly properties. In our previous study, nanoparticles were mainly observed in single decoction of GRR and co-decoction of GRR and PRA, but only few nanoparticles were formed in single decoction of PRA24 We speculated that nano-assemblies in SGD were mainly formed from the GRR components. The protein accounting for most of the weight of SGD-SAN was found to mainly come from GRR,36 and can self-assemble into nanostructures with functional capability to encapsulate active ingredients.19–22 Therefore, GP was extracted by an acetone precipitation method22 with a modification and was exploited to fabricate Pae GP-SAN. The extracted GP was characterized by SDS-PAGE, and the band of GP was located between 25–35 kDa (Figure 1C). In addition, GL was the main active ingredient and has been reported to form nanomicelles by self-assembly to improve the oral absorption of natural molecules and therapeutic agents.18,37–39 Therefore, Pae GL-SNM was fabricated via the film dispersion method, with a formulation consistent with that of our previous study.18
Characterization of Nano-Assemblies
The physicochemical properties of the three nano-assemblies are presented in Table 3 and Figure 2. Significant differences were observed in the physicochemical properties of the three nano-assemblies. The mean particle sizes of SGD-SAN, Pae GL-SNM, and Pae GP-SAN were around 130, 150 and 180 nm (Figure 2 A1-A3), respectively, and their PDI were 0.354, 0.167, and 0.154, respectively. The PDI value exceeding 0.3 for SGD-SAN reflects a broad particle size distribution, whereas both Pae GL-SNM and Pae GP-SAN exhibited PDI values below 0.2, suggesting a narrow size distribution.40 SGD-SAN exhibited the minimum particle size and maximum PDI, which may be due to its complex composition and sources. Repeated dialysis and centrifugation effectively removed larger particles, resulting in reduced particle size. The complex composition of SGD-SAN may contribute to its instability, leading to a broad particle size distribution. The TEM images demonstrated that SGD-SAN, Pae GL-SNM, and Pae GP-SAN were spherical in shape (Figure 2 B1-B3), with estimated average sizes of 50–300 nm for SGD-SAN and 100–200 nm for Pae GL-SNM and Pae GP-SAN, which is similar with the DLS measurements.
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Table 3 Physicochemical Properties of Nano-Assemblies (Mean ± SD, n=3)
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Figure 2 Particle size distribution and SEM images of Pae nano-formulations. (A1) and (B1) representing particle size distribution (PS:133 ± 13 nm, PDI:0.354 ± 0.015) and SEM images of SGD-SAN; (A2) and (B2) are particle size distribution (PS:154 ± 8 nm, PDI:0.167 ± 0.013) and SEM images of Pae GL-SNM; (A3) and (B3) are particle size distribution (PS:184 ± 7 nm, PDI:0.154 ± 0.009) and SEM images of Pae GP-SAN.
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Both SGD-SAN and Pae GP-SAN exhibited zeta potentials of approximately −15 mV, suggesting limited colloidal stability, whereas Pae GL-SNM demonstrated a substantially higher absolute zeta potential (−35.88 mV), which was associated with enhanced stability due to stronger electrostatic repulsion.18 The similar zeta potentials observed for SGD-SAN and Pae GP-SAN may be attributed to their shared protein constituents, which likely dominate the surface charge characteristics of these nanoparticles. The EE of SGD-SAN, Pae GL-SNM, and Pae GP-SAN were 44.07%, 47.94% and 36.56%, respectively. The relatively low EE (less than 50%) observed for SGD-SAN, Pae GL-SNM, and Pae GP-SAN could be attributed to the high hydrophilicity of Pae.41 The DL of SGD-SAN, Pae GL-SNM and Pae GP-SAN were 5.54%, 10.70%, and 21.52%, respectively. The discrepancies in DL among SGD-SAN, Pae GL-SNM, and Pae GP-SAN are likely due to differences in the structural composition and size of the nanoparticles. SGD-SAN exhibited the lowest DL of Pae, because it encapsulated not only Pae but also other active ingredients, such as albiflorin, benzoylpaeoniflorin, and liquiritin.
In Vitro Drug Release
The in vitro release profiles of SGD-SAN, Pae GL-SNM, and Pae GP-SAN are shown in Figure 3. Similar drug release profiles were observed for the same Pae formulations in both the SGF and SIF. Compared to the Pae solution, Pae release from the three nano-assemblies (SGD-SAN, Pae GL-SNM, and Pae GP-SAN) exhibited significantly slower kinetic profiles. About 100% of Pae was rapidly released from the Pae solution within 2 h, whereas only 50%-75% of Pae was released from SGD-SAN, Pae GL-SNM, and Pae GP-SAN in SGF and SIF, which is likely due to the sustained diffusion barrier imposed by the nano-assemblies matrix and drug-carrier interactions.42 In addition, the Pae release from three nano-assemblies within 6 h in SGF followed the order: Pae GP-SAN > SGD-SAN > Pae GL-SNM, with Pae GP-SAN exhibiting the fastest release kinetics (74.61% at 2 h vs 61.54% for SGD-SAN and 53.20% for Pae GP-SAN), likely due to protein hydrolysis of GP in an acidic environment, which compromises the structural integrity of nano-assemblies and promotes payload liberation. Pae GL-SNM exhibited a relatively lower cumulative Pae release (53.20%) in SGF than in SIF (64.11%), which may be due to the suppression of Pae GL-SNM disassembly under low pH conditions, thereby maintaining the structural integrity of micelles and delaying drug release. The sustained release of Pae from the nano-assemblies may be beneficial for the oral absorption of Pae by facilitating prolonged mucosal interactions.43
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Figure 3 In vitro release profiles of Pae from Pae solution, SGD-SAN, Pae GL-SNM and Pae GP-SAN in SGF (A) and SIF (B) (mean ± SD, n=3).
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Intestinal Absorption Characteristics of Nano-Assemblies
The intestinal perfusion studies in rats are the most reliable and cost-effective option among all methods for assessing intestinal drug absorption due to the high correlation between human and rat small intestinal permeability (r2 = 0.8–0.95).29,44 The in situ intestinal perfusion has been widely used to study the absorption of drugs in the intestine.18,28,29,39,45 Therefore, the absorptive behaviors of SGD-SAN, Pae GL-SNM, Pae GP-SAN, and Pae solution were compared by the in situ intestinal perfusion method, and gravimetry was employed instead of the classic phenol red method to determine perfusate volume changes in our study, owing to the partial intestinal absorption of phenol red.29
The Ka and Peff obtained from the single-pass intestinal perfusion models are shown in Figure 4. Pae solution showed similar Ka and Peff values in the jejunum and ileum. The Ka and Peff values of SGD-SAN, Pae GL-SNM and Pae GP-SAN were higher than those of the Pae solution, indicating increased intestinal absorption of Pae by nano-assemblies. This may be explained as follows: firstly, GL in SGD-SAN and Pae GL-SNM may reduce drug efflux through the inhibition of P-gp and increase drug penetration through the intestinal epithelium by increasing permeability and decreasing the elasticity modulus of the cell membranes.46,47 Secondly, the excellent mucoadhesion of nanoparticles to GI prolongs the drug residence time, possibly leading to increased drug absorption.48 Finally, internalization of nanoparticles by epithelial cells may also contribute to intestinal absorption of Pae.49 Among the three nano-assemblies, Pae GP-SAN showed the highest Ka and Peff values in the ileum, suggesting the best intestinal absorption. The possible explanation is as follows: a key distinction between the jejunum and the ileum lies in the presence of specialized structures such as Peyer’s patches (PPs) in the latter.50 The follicular-associated epithelial cells (FAE) are enriched in the PPs, where many membranous/microfold cells (M cells) reside in the FAE region.51 M cells are specialized antigen-transporting cells characterized by abundant vesicles, short microvilli, and low enzymatic activity, with a primary function of transporting antigens from the intestinal lumen to the subepithelial lymphoid tissue.52 Involved in antigen sampling, M cells have been extensively reported to possess strong transcytosis capacities to transport a wide range of particulate substances, including antigens, bacteria, viruses, and therapeutic nanoparticles. As an exogenous protein nanoparticle, Pae GP-SAN, with protein-rich composition, is likely to be recognized as antigenic substance and preferentially internalized by M cells, which likely accounts for its greater absorption in the ileum.49
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Figure 4 The
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Pharmacokinetics of Nano-Assemblies
The plasma concentration-time curves of Pae after oral administration of Pae solution, SGD-SAN, Pae GL-SNM, and Pae GP-SAN are shown in Figure 5, and the main pharmacokinetic parameters are presented in Table 4. In agreement with our previous findings,18 Pae solution demonstrated poor oral absorption, as evidenced by its low Cmax (964.89 ± 128.81 ng/mL) and AUC0-t (5676.14 ± 311.61 ng/mL·h), which could be attributed to its high hydrophilicity, P-gp mediated efflux, and hydrolysis in the intestine.53 Compared with the Pae solution, SGD-SAN exhibited a relatively higher plasma concentration of Pae at all time points following oral administration. The Cmax (1907.48 ± 245.22 ng/mL) and AUC0-t (8241.69 ± 944.62 ng/mL·h) of Pae after oral administration of SGD-SAN were significantly higher than those of Pae solution, indicating significant enhancement in the oral bioavailability of Pae. Furthermore, the results also suggested that the bioavailability improvement of Pae by oral administration of SGD may be related to the formation of SGD-SAN during decoction. The bioavailability improvement of Pae by SGD-SAN can be attributed to the prolonged drug release and the increased intestinal absorption. In addition, both the bioadhesive properties and the overall transmembrane capacity of nanoparticles also probably contribute to the enhanced Pae absorption.42,54
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Table 4 The Pharmacokinetic Parameters of Pae Following Oral Administration of Pae Solution, SGD-SAN, Pae GL-SNM and Pae GP-SAN (Mean ± SD, n=6)
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Figure 5 Plasma concentration-time curves of Pae following oral administration of Pae solution, SGD-SAN, Pae GL-SNM and Pae GP-SAN (mean ± SD, n=6).
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Notably, the bioavailability of Pae was also significantly improved by the oral administration of Pae GL-SNM and Pae GP-SAN, as evidenced by the higher AUC0-t (9362.52 ± 854.15 ng/mL·h for Pae GL-SNM and 11209.01 ± 2093.72 ng/mL·h for Pae GP-SAN). The same reason as proposed above with SGD-SAN can be employed to interpret the bioavailability improvement of Pae by the oral administration of Pae GL-SNM and Pae GP-SAN. Moreover, Pae GP-SAN showed significantly higher AUC0-t as compared to SGD-SAN, which could be explained by the increased intestinal absorption in the ileum. Both intestinal perfusion and pharmacokinetic studies demonstrated that SGD-SAN, Pae GL-SNM, and Pae GP-SAN significantly enhanced the oral absorption of Pae, with Pae GP-SAN exhibiting the best oral absorption. These results suggested that nano-assemblies, particularly protein-based GP-SAN, overcome the absorption barriers of Pae through ileum-targeted uptake, sustained release, and nanoparticle bioadhesion, providing a structural foundation for SGD’s bioavailability-enhancing effect.
Potential Formation Mechanisms of Nano-Assemblies
As is well known, urea and Tween 20 are capable of disrupting hydrogen bonds and hydrophobic interactions, respectively, while NaCl can reduce electrostatic interactions;20 these chemical interference agents have been widely used to study the mechanism of nanocomplex formation.33,34 To gain more insights into which dominant driving forces are present, we investigated the particle size change of SGD-SAN, Pae GP-SAN, and Pae GL-SNM after adding urea, NaCl, and Tween 20. As shown in Figure 6A, increasing concentrations of urea significantly increased the particle size of Pae GL-SNM, while exerting no effect on that of SGD-SAN and Pae GP-SAN, indicating that hydrogen bonds may not play a dominant role in driving the assembly or maintaining the structural stability of SGD-SAN and Pae GP-SAN, but play a non-negligible role in either the formation or, more notably, the structural stability of Pae GL-SNM.
 |
Figure 6 The particle size change of SGD-SAN, Pae GP-SAN, and Pae GL-SNM after adding urea (A), NaCl (B), and Tween 20 (C).
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Figure 6B showed that increasing concentrations of NaCl exerted no effect on the particle size of SGD-SAN, led to a slight increase in the particle size of Pae GP-SAN, and caused a significant increase in the particle size of Pae GL-SNM. Notably, when the NaCl concentration was equal to or higher than 0.05 M, Pae GL-SNM underwent obvious gelation. The absence of particle size change of SGD-SAN with increasing NaCl concentrations indicates that its stable dispersion is completely independent of electrostatic interactions. The slight particle size enlargement of Pae GP-SAN implies a weak reliance on electrostatic interactions. The significant particle size increase and subsequent gelation (at NaCl ≥ 0.05 M) of Pae GL-SNM demonstrate its strong dependence on electrostatic interactions.
Increasing concentrations of Tween 20 exerted a significant impact on the particle sizes of SGD-SAN, Pae GP-SAN, and Pae GL-SNM, but with distinct concentration-dependent trends (Figure 6C). The particle size increased significantly with rising Tween 20 concentration and tended to plateau (~800 nm) after the concentration reached 5 mM, while the particle size of Pae GP-SAN remained unchanged initially and then increased significantly (~1600 nm) when the Tween 20 concentration reached 10 mM. The results indicated that hydrophobic interactions play a critical role in maintaining the stability of SGD-SAN and Pae GP-SAN. For Pae GL-SNM, the particle size increased slightly to approximately 200 nm at Tween 20 concentrations below 2 mM, followed by a significant decrease to approximately 20 nm at and above 5 mM. This is because at low concentrations, Tween 20 only partially inserts into the Pae GL-SNM, causing a slight size increase due to structural loosening or shell expansion. However, once a critical concentration is exceeded, it sufficiently disrupts the hydrophobic interactions to disassemble the original micelles and form smaller mixed or Tween 20-dominated micelles, resulting in a significant size reduction.
Based on the above results, we can draw the following conclusions: hydrophobic interactions serve as the common core force across all nano-assemblies, while their dependencies on other intermolecular forces differ significantly. Specifically, hydrophobic interactions act as the core driving force for the formation of Pae GL-SNM, with hydrogen bonds and electrostatic interactions jointly assisting in maintaining the structural stability of the formed micelles; for Pae GP-SAN, its formation is driven by a synergistic effect dominated by hydrophobic interactions and supplemented by electrostatic interactions; while for SGD-SAN, hydrophobic interactions may alone constitute the core force supporting its structural stability.
Conclusions
In this study, three nano-assemblies of different components from SGD (SGD-SAN, Pae GL-SNM, and Pae GP-SAN) were compared in vitro and in vivo, with particular emphasis on their differential effects on Pae bioavailability. Three nano-assemblies showed similar spherical shape, but exhibited significant differences in particle size (133 nm for SGD-SAN, 154 nm for Pae GL-SNM, and 184 nm for Pae GP-SAN), PDI, zeta potential, EE, and DL (5.54% for SGD-SAN, 10.70% for Pae GL-SNM, and 21.52% for Pae GP-SAN). While hydrophobic interactions act as the common core force driving the formation of all nano-assemblies, their dependencies on other intermolecular forces vary remarkably. SGD-SAN, Pae GL-SNM, and Pae GP-SAN exhibited sustained Pae release (50–75% over 12 h vs 100% for the Pae solution in 2 h). In situ intestinal perfusion experiments in rats showed significantly higher effective permeability coefficients (Peff) for all nano-assemblies than the Pae solution, with Pae GP-SAN exhibiting the highest ileal absorption, which may be attributed to preferential M-cell uptake facilitated by its larger size and protein-rich composition. Pharmacokinetic studies further confirmed superior performance of Pae GP-SAN with the highest AUC0-t (11209.01 ± 2093.72 ng/mL·h) and Cmax (2896.04 ± 255.01 ng/mL), representing 2.0-fold and 3.0-fold increases over Pae solution (5676.14 ± 311.61 ng/mL·h and 964.89 ± 128.81 ng/mL), respectively. Additionally, Pae GL-SNM and SGD-SAN significantly enhanced Pae bioavailability, with AUC0-t increased by 65% and 45%, respectively. These results suggested that nano-assemblies, particularly protein-based Pae GP-SAN, overcome the absorption barriers of Pae through ileum-targeted uptake, sustained release, and nanoparticle bioadhesion, thereby providing a structural basis for SGD’s ability to enhance Pae bioavailability. The comprehensive comparisons elucidated the structure-function relationships underlying SGD-mediated improvements in Pae absorption. However, the reasons for the difference in Pae absorption among the three nano-assemblies remain unclear. In particular, no direct experimental evidence has been provided to support ileal M-cell uptake proposed as a key mechanism for Pae GP-SAN’s superior absorption. Further studies should be performed to clarify their mechanisms for enhancing the oral absorption of Pae.
Acknowledgments
This work was partially supported by the National Natural Science Foundation of China (82260848), the Natural Science Foundation of Jiangxi Province (20232BAB216139), – the Technology Innovation Team of Jiangxi University of Traditional Chinese Medicine (CXTD22006)-, and the Innovation Special Fund for graduate students of Jiangxi University of Chinese Medicine (202510412257).
Author Contributions
All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.
Disclosure
The authors have no relevant financial or non-financial interests to disclose.
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