Enhanced Anti-inflammatory Effect of Puerarin through Optimized Release and Bioavailability via PDLG-Loaded Nanoparticles

Abstract

Puerarin (PU), a bioactive constituent reported to possess therapeutic effectiveness, but it suffers a drawback of poor bioavailability. In the present study, the PU nanoparticles (PU-NPs) were prepared using solvent-diffusion-evaporation method and optimized using Box–Behnken design (BBD), a response surface methodology for obtaining the optimal material ratio of PU-NPs. Further, PU and PU-NPs were evaluated to assess their cytotoxic effect and in vitro efficiency of inflammatory responses using lipopolysaccharide-sensitive macrophage cell line (RAW264.7). Also, PU-NPs were assessed for, in vivo anti-inflammatory activity using a carrageenan-induced rat paw edema model and an oral pharmacokinetic release study. PU-NPs formulation exhibited smaller particle sizes, an increase in the amorphous structure stability, and a higher dissolution rate, as compared to PU. The relative bioavailability of PU-NPs increased up to five-fold compared to PU suspension, as demonstrated by the parameters like the area under the curve (AUC), t_1/2, and the mean residence time (MRT). It mitigates enhanced cell viability and lowers the production of pro-inflammatory mediators [nitric oxide (NO), tumour necrosis factor-α (TNF-α), and interleukin-6 (IL-6)]. Moreover, PU-NPs showed a marked reduction in the development of paw edema at low doses compared to PU in an in vivo carrageenan-induced rat paw edema model. The results of the study affirm the potential of PU-NPs compared to PU in enhancing in vitro and in vivo anti-inflammatory responses by prolonging release and enhancing relative bioavailability.

INTRODUCTION

Pueraria tuberosa (Willd.) (Indian Kudzu root) is a perennial herb commonly known as ‘vidarikanda,’ distributed throughout India, especially in the southeast part.^1,2) The tubers of P. tuberosa are widely used in ethnomedicine as well as in traditional systems of medicine, particularly in Ayurveda. Puerarin (PU) is a major bioactive constituent of Pueraria tuberosa with highly edible and health values, commonly used as an adjunctive treatment for inflammatory diseases, diarrhoea, and fever in Ayurvedic and traditional Chinese medicine.^1,3) The therapeutic effectiveness of PU is constrained by its poor water solubility (2.58 g/L)⁴⁾ and low bioavailability; hence, according to the Biopharmaceutics Classification System (BCS), it is categorized as a Class IV drug.^4,5) In the pharmaceutical sciences, the development of novel drug technologies such as synthetic water-soluble prodrugs, anionic polymerization, phospholipid complexes, cyclodextrin inclusion, microemulsions, and solid lipid nanoparticles for the delivery of drugs with poor solubility and permeability attracts more attention. Nanoparticles are a unique approach with their small size and high surface area-to-volume ratio, which makes them highly efficient drug delivery technologies.^6–8) There are various nano-formulations reported previously to overcome the poor bioavailability of PU (>3%), like nano-emulsions using co-solvent approach followed by freeze drying,⁹⁾ nanoparticles using different polymers, and formulation methods,^6,8,10,11) which help in enhancing the bioavailability of PU.

Inflammation is a prominent event in most acute as well as chronic disorders and involves numerous cells, a complicated network of mediators, and the activation of several pathways. Chronic inflammation causes respiratory, autoimmune, and cardiovascular disorders, as well as rheumatoid arthritis, diabetes, cancer, Alzheimer’s disease, and atherosclerosis.¹²⁾ Furthermore, macrophages are a type of white blood cell that is crucial for immune responses that shield the host against microbial invasion and tissue damage and is also essential for the regulation of inflammation. During an inflammatory process, activated macrophages elicit an array of functional responses, including the production of cytokines (e.g., tumor necrosis factor (TNF) and interleukin (IL)) and nitric oxide (NO).^12,13) In addition to the study of anti-inflammatory activity, carrageenan-induced rat paw edema is a well-established animal model widely used to screen novel anti-inflammatory compounds by causing acute biphasic edema in the rat paw.^14,15) PU showed anti-inflammatory activity in various inflammatory diseases, viz., osteoarthritis, depression, intracerebral haemorrhage, Alzheimer’s disease, respiratory disease, and lung injury.³⁾ The detailed study on the preparation and characterization of PU nanoparticles has been reported by us.¹⁶⁾ This study intends to investigate the in vitro (using RAW264.7 macrophages cell line) and in vivo (carrageenan-induced rat paw edema model) anti-inflammatory activity of nanoparticles, with an emphasis on the significant improvement in their bioavailability.

MATERIALS AND METHODS

Materials

Puerarin (purity = 97.2%) was isolated from P. tuberosa rhizomes and used in the study. Polymer PDLG 5002A (50/50 DL-lactic/glycolide copolymer) was a gift sample procured from Corbion, Purac–Biochem BV Netherlands. Surfactant Pluronic F₁₂₇, ibuprofen, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lipopolysaccharide (LPS), Griess reagent, and carrageenan were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). The RAW264.7 macrophage cell line was obtained from the National Centre for Cell Science (NCCS) in Pune and grown in Dulbecco’s modified Eagle’s medium (DMEM, Hi-Media, India) supplemented with 10% fetal bovine serum (FBS; Hi-Media, India) and antibiotics (Hi-Media). Enzyme-linked immunosorbent assay (ELISA) kits for tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were purchased from Elabscience, U.S.A. All other reagents used were of analytical reagent grade for the present study.

Development of PU-NPs

PU-NPs were prepared using an emulsion solvent evaporation method, followed by lyophilization.^17–19) Briefly, a flow chart of the process to prepare PU-NPs is shown in Fig. 1. The organic phase was prepared using 100 mg of PDLG polymer (equivalent to 5.5% wt.), and 4 mg of PU was dissolved in 1.8 mL of ethyl acetate and ethanol at 30% v/v (co-emulsifiers), resulting in a translucent and low-viscous solution. The aqueous phase (surfactant solution) was prepared by mixing Pluronic F₁₂₇ (0.2 g) equivalent to 2% wt in 10 mL of water. The organic phase is then dropwise added to the aqueous phase with continued stirring on a magnetic stirrer for 20 min at 1000 × g, resulting in the formation of an O/W pre-emulsion. Through employing probe sonication at an amplitude of 30 m for 2 min with a pulse sequence of 30 s, the formulated O/W pre-emulsion was reduced. In order to diffuse and evaporate the organic phase, the O/W primary emulsion was added to 10 mL of water while being constantly stirred. This led to nanoprecipitation and the formation of a colloidal suspension. The suspended nanoparticles were centrifuged at 10000 × g, −5 °C for 20 min and washed repeatedly with water to remove excess surfactant, and lyophilized to obtain powder.¹⁷⁾

Fig. 1. The Flow Chart of Excremental Process for PU-NPs Formulation by Solvent Evaporation Diffusion Method

Cell Culture

The murine macrophage cell line, RAW264.7 cells (established from male mice induced with Abelson murine leukemia virus), was obtained from the National Centre for Cell Science (NCCS) in Pune. These cells are chosen as an in vitro model because they produce a robust and well-known anti-inflammatory response, especially when challenged by inflammatory stimulants such as LPS, NO, and cyanine.^20,21) The cells were cultured in DMEM containing 10% fetal bovine serum and antibiotics, and the cells were incubated in a humified incubator at 37 °C under 5% CO₂, and the media was replaced with fresh media every two days. Cytotoxicity and nitric oxide assays were performed.

MTT Cytotoxicity Assay

Study was assessed using the MTT assay. In brief, a 96-well plate was seeded with the RAW264.7 murine macrophage cell line at a density of 2 × 10⁵ cells per well in DMEM media and incubated overnight. MTT assay was performed to ensure that the observed NO inhibition by PU-NPs is not due to cytotoxicity. PU and PU-NPs at different concentrations (2, 5, 20, and 50 µg/mL) and LPS (500 ng/mL) were dissolved in DMEM and replaced with medium during treatment and incubated for an additional 24 h. After 24 h of incubation with the treatments, 10 µL of a 5 mg/mL MTT solution was added to each well and incubated for an additional 4 h at 37 °C. After that, the crystal was left at the bottom of the plate, and the supernatant was discarded. The insoluble formazan salt was then dissolved by adding 100 µL of dimethyl sulfoxide, and the plate was incubated for another twenty minutes. A microplate reader with an absorbance value of 570 nm was used to quantify the formazan formed.^16,21–23)

Nitric Oxide Assay

96-well culture plates were seeded with 2 × 10⁵ RAW264.7 cells per well. PU and PU-NPs (5 and 20 µg/mL) were added to the cells before they were incubated for an hour. LPS (500 ng/mL) was used in order to stimulate cells, while they were subsequently kept in a CO₂ incubator at 37 °C for an additional 24 h. Using a modified Griess reagent, NO release in culture media was ascertained in compliance as per manufacturing protocol. The cell supernatant was mixed with an equal volume of modified Griess reagent solution, and absorbance was measured at 540 nm using envision microplate reader (PerkinElmer, Inc., Waltham, MA, U.S.A.).^23–25)

Inflammatory Cytokine Immunoassay

TNF-α and IL-6 production of inflammatory cytokines was measured in response to PU and prepared PU-NPs. The cells were grown and treated to comply with the preceding section’s instructions. Following the manufacturer’s instructions, specialized ELISA kits were used to test the supernatant that was taken from the wells of a 96-well plate.^25,26)

Animals

The experiment was carried out in compliance with the guidelines of the Committee for Control and Supervision of Experiments on Animals (CCSEA), Government of India, on animal experimentation. Male Wistar rats weighing between 100–150 g were purchased from the Central Animal House facility of Panjab University, Chandigarh, India. The Institutional Animal Ethical Committee (CCP/IAEC/Feb 22/7), CCP, Landran, Mohali, India, approved the experimental protocol. A week before the experiment, the rats were placed in an environmentally controlled room with a temperature 25 ± 2 °C, humidity 60 ± 5%, 12-h dark/light cycle. Water was provided to them freely, along with a standard pallet diet.

Carrageenan Induced Anti-inflammatory Activity

The anti-inflammatory activity of PU and PU-NPs was evaluated using the carrageenan-induced paw edema method.^27,28) Rats were fasted overnight with free access to water prior to the start of the experiment and divided into seven groups (n = 6). The normal control group was treated with vehicle (normal saline) only; the positive control group was treated with reference drug, indomethacin at a dose of 12.5 mg/kg per oral; the negative control was treated with 100 µL of 1% carrageenan in saline solution; and the remaining four groups were treated with PU at 12.5 mg/kg and nanoparticles (PU-NPs) at 7.5, 10, and 12.5 mg/kg (The amount of PU in 12.5, 7.5, and 10 mg of nanoparticles was found to be 1.81, 1.45, and 1.01 mg, respectively as per drug loading in nanoparticles) orally. Rats’ left hind paws were subcutaneously injected with 100 µL of 1% carrageenan in a saline solution in order to cause edema an hour after the PU and PU-NPs of different doses were administered. Using a plethysmometer, the paw volume was measured immediately (basal) and three and five hours following the carrageenan injection. By deducting the initial paw volume from the paw volume obtained at the 3 and 5-h time intervals, edema was calculated. The following formula was used to calculate the mean difference in paw volume while determining the percentage of inhibition:

  

Where,

• Vc = mean paw volume of control group (mL),
• Vt = mean paw volume of test group (mL),

Pharmacokinetic Study

The pharmacokinetics study of PU and PU-NPs was performed on Wistar rats (100–150 g).^29,30) Animals were fasted overnight before the experiment with free access to water, and then twelve rats were divided into two groups. The first group of animals was administered PU suspension (12.5 mg/kg), and the other group was administered PU-NPs (12.5 mg/kg, most active anti-inflammatory dose) suspended in water orally to determine and directly compare the release of puerarin from nanoparticles to free puerarin in suspension form. A subclavian vein blood sample of 0.3 mL was taken at 0 min, 10 min, 30 min, 1, 2, 4, 6, 8, 12, 24, and 48 h. Samples were collected in heparinized tubes, and plasma was separated instantly by centrifuging at 13000 × g for ten minutes. Before analysis, the collected plasma was preserved at −20 °C.

HPLC Analysis

The extraction of PU from the plasma sample was conducted as follows: 20 µL of internal standard solution (apigenin 0.5 mg/mL), 75 µL of methanol-ethyl acetate (90 : 10 v/v) solution, and 100 µL of methanol-acetonitrile (90 : 10 v/v) were added to 100 µL of plasma sample, vortexed for 5 min, and centrifuged at 10000 × g for 10 min. The supernatant was separated and subjected to analytical HPLC analysis employing Waters HPLC system, which was equipped with an in-line degasser, Delta 600 quaternary solvent pump, 2998 photodiode array (PDA) detector, 2707 Plus autosampler, Breeze 2 software (Waters, Milford, MA, U.S.A.), and a temperature control module. For the chromatographic separation, an RP-18 X-Bridge column (4.6 × 250 mm, 5 µm) was implemented. Gradient elution was used throughout the process of separation, 0.1% formic acid in water (solvent A) and methanol (solvent C) eluted in a gradient manner (0–5 min 20% C; 5–10 min 25% C; 10–15 min 30% C; 15–20 min 45% C; 20–21 min 55% C; 21–25 min 85% C; 25–27 min 95% C, 27–30 min 95% C, 30–33 min 20% C) and a flow rate of 1 mL/min. Peaks were monitored using a PDA detector at 254 nm.

Pharmacokinetic Study Parameters

The pharmacokinetic parameters for PU in plasma were estimated by appropriate compartmental models. All parameters were determined from the sample collection times and the assayed concentrations at these times. Concentration values below the lower limit of quantification were set to zero. Plasma PU concentrations were plotted against time and the pharmacokinetic calculations were performed using the standard software PK solver 2.0. The area under the curve (AUC) of the plasma drug concentration to time from 0 to 24 h and its extrapolation area were determined by the linear trapezoidal rule. The maximum PU concentration in plasma (C_max) and time taken to reach the maximum plasma concentration (t_max) were obtained from the plasma data. Kel represents the apparent terminal rate constant and it was calculated by the linear regression of the log-concentrations of the drug in the terminal phase. The half-life (t_1/2) of the terminal elimination phase was calculated using formula t_1/2 = 0.693/Kel.

The other pharmacokinetic parameters were also estimated like mean residence time (MRT) of PU was calculated using trapezoid area calculations extrapolated to infinity

  

Where:

• AUMC: The total area under the first moment of drug concentration curve from zero to infinity (determined by combining trapezoid calculation of AUMC (0–24) and its extrapolated area)

total plasma clearance (CL), by using the following formula

  

Where:

• CL: is the total clearance (typically in units of mL/min or L/h).
• Dose_oral: is the oral dose of the drug.
• F: is the bioavailability
• AUC_0–∞: is the area under the plasma concentration–time curve from time zero to infinity after the oral dose.

The relative bioavailability (F) of PU-NPs was calculated using following formula

  

where:

• AUC_test: the area under the concentration–time curve for the test formulation.
• AUC_reference: the area under the concentration–time curve for the reference formulation.

The student’s t-test was used to analyse differences between two groups. The difference in two groups of data with p-value of less than 0.05 or 0.01 was considered significant. The data were presented as mean ± standard deviation (S.D.).^31–33)

Method Validation

Following the bioanalytical method validation (U.S. Food and Drug Administration (FDA), 2001), the analytical method was validated to demonstrate lower limit of detection (LLOD), lower limit of detection (LLOQ), recovery, linearity, robustness, accuracy and precision, Specificity was confirmed by peaks at the retention times for both puerarin and the internal standard. Recovery was evaluated by comparing the peak areas from extracted plasma with those obtained by directly injecting spiked standard solutions into a methanol solution. Three concentrations of puerarin were analysed in triplicate (50, 500, and 1000 ng/mL in plasma samples). The LLOQ and LLOD were calculated based on a signal-to-noise ratio of 10 : 1 and 3 : 1, respectively.³⁴⁾

Linearity was assessed across eight concentration levels, ranging from 10–2000 ng/mL (10, 40, 80, 200, 400, 800, 1200, and 2000 ng/mL) in plasma samples. Calibration curves were generated by plotting the ratio of the peak area of puerarin to that of the internal standard (apigenin) against the concentration of puerarin. The slope, intercept, and correlation coefficient were calculated using linear least-squares regression analysis. A robustness study was conducted to assess the method’s reliability and consistency under varying conditions that could arise during routine application. The evaluation involved altering key parameters, including pH levels (0.1, 0.2, and 0.05) and flow rates (1, 0.9, and 1.1 mL/min). This was performed using three sample concentrations (50, 500, and 1500 ng), each analysed in triplicate.³⁵⁾

Accuracy and precision were evaluated through six replicate samples of puerarin at concentrations (low, medium, and high concentrations) of 50, 500, and 1500 ng/mL in blank rat plasma samples, processed and analysed over three consecutive days. Accuracy was calculated by comparing the measured concentration to the known spiked concentration, based on calibration curves. Intra-day and inter-day precision were measured by calculating the relative standard deviation (RSD). The average precision (RSD at each concentration) should fall within 15% of the nominal value, except at the lower limit of quantification (LLOQ), where a deviation of no more than 20% is acceptable.^36,37)

Statistical Analysis

The anti-inflammatory activity data was calculated as mean ± standard error of the mean (S.E.M.) following the pharmacokinetic data, which was analyzed by Student’s t-test for unpaired observations compared with the control, and the (*) significance of difference among the various test, * p < 0.05, ** p < 0.01, and *** p < 0.001 were considered as statistically significant. The statistical software GraphPad Prism was utilized for all statistical analyses.

RESULTS

Characterization pf PU-NPs

PU-NPs were prepared using an optimization study and evaluated by using different parameters. The particle size was found to be 120.6 ± 0.03 nm with a PDI of 0.22. The zeta potential of the PU-NPs measured by employing a Delsa nano zeta-sizer at 25 °C was found to be −16.3 mV. The encapsulation efficiency, drug loading, and drug content of the PU-NPs were reported at 90.21, 14.56, and 98%, respectively. Based on the findings from FTIR data, PU was only encapsulated in PDLG by possible intermolecular forces such as hydrogen bonds and did not take part in the polymerization reaction. Additionally, the amorphous shape of PU-NPs, as revealed by X-ray diffraction (XRD) data, makes them more stable than PU.¹⁶⁾

Cell Viability Study

The cell viability of the control group (untreated) was designated at 100%, indicating no cytotoxicity. Test samples of PU and PU-NPs at concentrations of 2, 5, 20, and 50 µg/mL did not affect cell viability and showed cell viability of 89–100% at all concentrations with respect to the control. The graph of the cell viability of PU and PU-NPs with respect to the standard is shown in Fig. 2.

Fig. 2. The Effect of Four Different Concentrations of PU and PU-NPs (2, 5, 20, and 50 µg/mL) on the Viability of RAW264.7 Cells

Cytotoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay after 24 h treatment with both PU and PU-NPs and values are presented as the mean of three independent experiments in triplicate, and data are shown as mean ± standard deviation. Data are expressed as mean ± S.E.M. ^# p < 0.01, compared with normal control group; * p < 0.05, ** p < 0.01, compared with LPS group.

Nitric Oxide Assay

After incubating RAW264.7 macrophages with LPS for 24 h, an evident rise in NO production was observed in the culture medium. NO production was inhibited after treatment of macrophages with PU 5, 20 µg/mL and PU-NPs 5, 20 µg/mL. However, PU-NPs demonstrated the greatest and most significant inhibition at 20 µg/mL. Furthermore, as illustrated in Fig. 3, statistical analysis demonstrated that the PU-NPs concentrations significantly reduced NO production as compared to PU and LPS production.

Fig. 3. Effects of PU and PU-NPs on Lipopolysaccharide-Induced Nitric Oxide Production in RAW264.7 Macrophage

Cells were treated with two different concentrations of PU and PU-NPs (5 and 20 µg/mL) in the presence of 500 ng/mL lipopolysaccharide for 24 h. Dexamethasone (Dexa20 µg/mL) was used as a positive control. Results are expressed as means and standard deviation of three experiments in triplicate. (*) Indicates significance statistically difference compared to LPS group by * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001, respectively and ^# p < 0.01, compared with normal control group (unpaired Student’s t-test).

Inflammatory Cytokine Immunoassay

Figure 4 illustrates the anti-inflammatory activity of PU-NPs and PU in pro-inflammatory cytokines, TNF-α (a marker for pro-inflammatory cytokine activity) and IL-6 (a marker of anti-inflammatory activity) in the treated RAW264.7 macrophages. The findings demonstrate that the treatment of these cells with PU-NPs significantly decreased the production of TNF-α and IL-6 at the concentration of 20 pg/mL. Additionally, as shown in Fig. 4, statistical analysis revealed that PU-NPs significantly reduced TNF-α and IL-6 as compared to PU. So, PU-NPs showed a significant effect on both biomarkers, which helps minimize the dose of the drug.

Fig. 4. Effect of PU and PU-NPs (5 and 20 µg/mL) on the Production of Inflammatory Cytokines [A] Interlukin-6 (IL-6), and [B] Tumor Necrosis Factor-α (TNF-α) in LPS-Stimulated RAW264.7

Macrophages were stimulated with LPS (500 ng/mL) for 24 h. The concentration of cytokines in the supernatants was determined by ELISA. Dexamethasone (Dexa20 µg/mL) was used as a positive control. Results are expressed as means and standard deviation of three experiments in triplicate. *Significant difference (p < 0.001) when PU-NPs (5 and 20 pg/mL) compared to control group and #significant difference (p < 0.005) when PU-NPs (5 and 20 pg/mL) compared to PU.

Anti-inflammatory Activity

Anti-inflammatory activity of PU and PU-NPs were also studied using carrageenan-induced rat paw edema model. Typical macroscopic photographs of the rat’s right paw edema shown in Fig. 5. Compared to PU, all PU-NPs concentrations reduce edema; Fig. 5 illustrates the time-dependent increase of paw edema after carrageenan injection. PU-NPs at 12.5 mg/kg dose showed a drastic reduction in swelling compared to PU as well as indomethacin. The PU-NPs exhibited a more potent anti-inflammatory (51.44% at 12.5 mg/kg per os (p.o.)) effect as compared to the PU (43.14% at 12.5 mg/kg p.o.) and indomethacin (48.10% at 12.5 mg/kg p.o.).

Fig. 5. [A] Effect of PU-NPs on Carrageenan-Induced Paw Edema in Rats

Representative macroscopic photographs of paw from the (A) control, (B) 1% carrageenan (CRG), (C) 1% carrageenan + indomethacin (CRG + IND), (D) 1% carrageenan + puerarin (CRG + PU), (E) 1% carrageenan + PU-NPs 7.5 mg/kg (CRG + PU-NPsL), (F) 1% carrageenan + PU-NPs 10 mg/kg (CRG + PU-NPsM), (G) 1% carrageenan + PU-NPs 12.5 mg/kg (CRG + PU-NPsH) given orally. Time course progression of rat paw edema model and the area under curve (AUC) and % reduced paw edema represented in graphs. Progression of *significant difference (p < 0.05), when PU-NPs (7.7, 10 and 12.5 mg/kg) compared to PU, and #significant difference (p < 0.05) when PU-NPs (12.5 mg/kg) compared to indomethacin.

Method Validation

The resulting chromatograms showed minimal endogenous interferences. Figure 6 shows representative chromatograms, including a blank plasma sample and a plasma sample collected 1 h after administering 12.5 mg/kg of PU and PU-NPs via oral dosing. The retention times for PU and the internal standard (apigenin) were approximately 11.66 and 21.87 min, respectively. Both peaks exhibited good shape, making them appropriate for accurate quantitative analysis.

Fig. 6. [A] Chromatogram of a Blank Plasma Sample, Showing No Endogenous Interferences, [B] Chromatogram of a Plasma Sample Collected 2 h after Oral Administration of 12.5 mg/kg of PU, [C] Chromatogram of a Plasma Sample Collected 2 h after Oral Administration of 12.5 mg/kg of PU-NPs

The retention times for PU and the internal standard (apigenin) were approximately 11.666 and 21.874 min, respectively.

The linear regression analysis yielded the equation y = 0.0104x + 0.1941, R² = 0.9997, with correlation coefficients for the calibration curves of all sample types exceeding 0.99, as shown in Fig. 7, demonstrating strong linearity. Puerarin recoveries from plasma samples at different concentrations (mean ± S.D., n = 3) were evaluated. The LLOQ and LLOD was calculated as 1.0 and 0.3 ng/mL, respectively. The low concentration corresponded to 50 ng/mL, the high concentration to 500 ng/mL of puerarin in plasma, and they were found 88.53 ± 0.07, and 89.82 ± 0.51%, respectively, confirming that the sample preparation approach was effective and suitable for accurate quantification of puerarin in rat plasma. The precision and accuracy results, as outlined in Table 1, demonstrate that the method was reliable and reproducible for quantifying puerarin in rat plasma. The robustness of the analytical method, was varied using two critical parameters, pH of the mobile phase (percentage of the formic acid) and flow rate of the chromatographic system. Despite these variations, the peak area for puerarin remained consistent across all experiments, indicating that the method is not significantly affected by minor fluctuations in pH and flow rate. These results (Table 2) demonstrate the robustness of the method, confirming its stability and reliability for the determination of puerarin in plasma samples.^38,39)

Fig. 7. Calibration Curve of PU

Table 1. Precision and Accuracy of the Method for the Determination of Puerarin in Rat Plasma (n = 3)

Sr. No.	Concentration (ng/mL)	Intraday		Interday
		Precision RSD%	Accuracy (%)	Precision RSD%	Accuracy (%)
1	50	5.80	97.71	2.43	95.33
2	500	2.01	96.29	1.81	97.51
3	1500	1.04	92.80	3.21	93.49

Table 2. Robustness Expressed in Percent Mean Recovery and Percent Relative Standard Deviation (n = 3)

Sr. No.	Parameter	Concentrations (ng/mL)	Retention time	Recovery (%)	% RSD
1	Mobile phase (A: water formic acid (0.1%))	60	11.666	100	1.57
		80	11.667	100	2.15
		1200	11.661	100	2.18
2	Mobile phase (A: water formic acid (0.05%))	60	11.731	97.86	4.90
		80	11.730	99.04	1.75
		1200	11.761	98.41	4.51
3	Mobile phase (A: water formic acid (0.2%))	60	11.691	100	6.16
		80	11.661	96.29	5.73
		1200	11.690	99.36	3.15
4	Flow rate (1 mL/min)	60	11.660	100	2.25
		80	11.668	100	6.35
		1200	11.666	100	5.06
5	Flow rate (0.9 mL/min)	60	11.631	96.71	1.33
		80	11.680	86.69	3.30
		1200	11.761	95.12	2.55
6	Flow rate (1.1 mL/min)	60	11.740	97.71	4.38
		80	11.700	92.63	5.64
		1200	11.766	96.95	3.46

Pharmacokinetic Data Analysis

PU’s poor oral bioavailability is primarily attributed to its low water solubility and limited intestinal membrane permeability. The use of PDLG nano-formulation could potentially be applied to increase the bioavailability of hydrophobic polyphenols and enhance the pharmacological actions. In this study, PDLG nanoparticles were incorporated into the oral formulation of PU to investigate their effect on bioavailability, given their proven success in enhancing the absorption of various natural substances, including daidzein by D-PNPs and D-CNPs (nanoparticles),²³⁾ curcumin by CUR-PLGA-NPs (nanoparticles).⁴⁾ The HPLC method was used to determine the mean concentration of PU in the plasma following the oral administration of a single dose in rats over a 24 h period. The retention times of PU and apigenin were 11.66 and 21.87 min, respectively showed in Fig. 6.^40,41) The mean plasma concentration–time profiles of PU after oral administration at a dose of 12.5 mg/kg for both PU and PU-NPs are shown in Fig. 8, and the pharmacokinetic parameters calculated from the data are summarized in Table 3. The concentration time profile was best described using a non-compartment model, following oral administration the plasma PU concentration increases sharply, C_max value for PU-NPs was approximately a 4.16-fold increase compared to the drug. Formulation (4 ± 0.29 h) has a 1.5-fold decrease in t_max compared to the drug (6 ± 0.11 h) and significantly increased, indicate slower and longer release of PU in the form of nanoparticles leads to prolonged drug action. The extend of the PU absorption was greater for the complex as seen from the significantly increased AUC_0–t values, rising from 938.89 ± 0.04* µg/mL h to 1050.75 ± 0.025* µg/mL h, (12% increase) p < 0.05. The AUC_0–∞ values showed that by formulation as a nanoparticle, the bioavailability of PU in rats was 11.08-fold higher, shows considerably better systemic drug exposure over time. This suggests that PU-NPs can provide extended therapeutic effects and significantly improve the efficiency of the drug delivery. t_1/2 from 2.55 ± 0.03h to 4.77 ± 0.16* h (1.87-fold increase) and MRT_0–t from 6.73 ± 0.008 h to 11.00 ± 0.030** h (1.24-fold enhanced) were prolonged respectively (p < 0.05) as compared to the PU suspension, although CL was reduced (5-fold) from 5.80 ± 0.21 mg/min to 1.16 ± 0.24* mg/min (p < 0.05) in the event of PU-NPs, a reduction in clearance indicates that the drug is eliminated more slowly from the body. This reduced clearance helps maintain therapeutic drug levels for a longer period, potentially leading to longer-lasting effects and reducing the need for frequent dosing.

Fig. 8. The Mean Plasma Levels of Puerarin versus Time after Single Oral Dose Administration of Puerarin as Suspension and Prepared Nanoparticles

The administered dose was 12.5 mg/kg in rats (n = 6).

Table 3. Comparative Pharmacokinetic Parameters Study of PU and PU-NPs, after Oral Administration

Pharmacokinetic parameters	Unit	Puerarin nanoparticles (PU-NPs)	Puerarin suspension (PU)
t1/2	h	4.77 ± 0.16**	2.55 ± 0.03
Tmax	h	6 ± 0.29*	4 ± 0.11
AUC0–t	µg/mL	1050.75 ± 0.025**	938.89 ± 0.04
Cmax	µg.h/g	1993.79 ± 0.014*	479.70 ± 0.08
AUC0–∞	µg.h/g	10875.02 ± 0.02**	980.96 ± 0.01
MRT0–t	h	7.77 ± 0.030*	6.25 ± 0.08
Cl	mg/min	1.16 ± 0.24*	5.80 ± 0.21

AUC_0–t, area under the concentration time curve from time zero to the last sampling time point; AUC_0–∞,, area under the concentration time curve from time zero to the infinity; C_max, maximum plasma concentration of PU; CL, total clearance; MRT, mean residence time; T_max, time of maximum plasma concentration of PU. **Comparison between two groups, p < 0.01, *Comparison between two groups, p < 0.01.

The relative bioavailability of the PU-NPs is approximately 561.22% compared to the PU, this means the PU-NPs has about 5.61 times more bioavailability of the PU. All parameter indicates that the absorption time of PU-NPs has been increased with improved GI retention time and elimination rate slowed when compared with PU. This could be due to the fact that PDLG nano-formulations have the potential to enhance the bioavailability of polyphenols. Encapsulating hydrophobic drugs in PLGA polymer represents a promising method for sustained and controlled drug delivery, significantly improving the bioavailability of BCS class IV drugs.⁷⁾

On the one hand, nanoscale particles are easily absorbed into the folds of the intestinal wall, while larger particles, due to their increased surface area relative to their volume, tend to dissolve more rapidly.⁹⁾ Conversely, nanoparticles designed for oral delivery can protect the drug from low pH environments, enhance absorption in the intestinal tract, and avoid undesired metabolic degradation. Additionally, the degradation characteristics of the carrier ensure sustained drug release. Furthermore, the amorphous dispersion of PU-NPs could also contribute to improved absorption.⁶⁾ Therefore, the pharmacokinetic studies provided the first proof of concept that PU-NPs may be suitable as long circulation and long-acting formulations of PU.

DISCUSSION

Novel drug delivery systems such as, phospholipid complexes, nanoparticles, and nanocrystals, have been reported to improve oral bioavailability of PU. The bioavailability of PU has increased up to 1.46-fold compared to oral suspension and other novel formulations of PU previously reported.^6,18,40) In the present study, we formulated PDLG-based PU-NPs for oral administration by the solvent diffusion evaporation method. When PU was incorporated into PU-NPs, the relative bioavailability of PU increased significantly. Further, the AUC, t_1/2, and MRT were also enhanced significantly as compared to PU suspension, while CL decreased markedly. PU-NPs were prepared using an optimization study and evaluated using different parameters, such as the particle size, PDI, zeta potential, encapsulation efficiency, drug loading, and drug content, FTIR analysis, XRD, in vitro release results indicated increased surface area of the nanoparticles likely facilitates better interaction with the gastrointestinal mucosa, enhancing absorption and release of the drug. Additionally, the nanoparticles’ small size might enable more efficient cellular uptake, contributing to the observed anti-inflammatory effects in the carrageenan model.

Inflammation is the body’s response to harmful stimuli, involving the activation of immune cells and the release of pro-inflammatory mediators like cytokines (TNF-α, IL-1β) and enzymes (cyclooxygenase (COX)-2). The carrageenan-induced rat paw edema model, which mimics acute inflammation, involves injecting carrageenan into the rat’s paw to induce swelling.⁴²⁾ Previous studies have reported that PU dose-dependently inhibited the production of pro-inflammatory cytokines IL-6 and TNF-α, as well as NO highlights its potential as a natural anti-inflammatory agent. Most significant inhibition of the IL-6, TNF-α, and NO pretreatment with PU was found at the concentration of 40 µM. Additionally, it was also reported that administering PU at a dose of 50 mg/kg resulted in a significant reduction of carrageenan-induced rat paw edema, a widely used experimental model for acute inflammation.^43,44)

In the present investigation, the prepared PU-NPs significantly showed enhanced bioavailability compared to PU and significantly reduced production of major pro-inflammatory mediators such as NO, TNF-α, and IL-6. Moreover, PU-NPs showed a very prominent anti-inflammatory effect in a carrageenan-induced rat paw edema model at a lower dose compared to the positive control indomethacin and PU. In the pharmacokinetic study, orally administered PU-NPs demonstrated a shorter t_max and rate of clearance. PU-NPs showed enhanced t_1/2, AUC_0–t, AUC_0–∞, C_max, and MRT with 4.4, 5.6, 4.2, and 1.63-fold effects, respectively. Finally, these data collectively support the idea that nanoparticle formulation is a promising drug delivery system for enhancing the oral bioavailability of PU. These findings suggest that our formulation could be a promising drug therapeutic approach for treating inflammatory conditions. The improved bioavailability allows for lower dosages. PU is a promising candidate for treating inflammatory conditions, but its low bioavailability limits direct use. Our findings suggest that PU-NPs could efficiently reduce inflammation at relatively low doses, highlighting their potential advantage over PU and traditional non-steroidal anti-inflammatory drugs (NSAIDs), which are associated with significant side effects such as gastrointestinal irritation and increased cardiovascular risk. Overall, these enhancements in pharmacokinetics may allow reduced dosing frequency, lower doses needed to achieve the same or better therapeutic outcomes, and prolonged action of the drug, making the nanoparticle formulation more efficient and effective than the suspension.

CONCLUSION

We found that PU and PU-NPs both possess anti-inflammatory effects. However, because of poor bioavailability, PU showed biological effects at higher doses. Comparatively, PU-NPs showed significant in vitro and in vivo anti-inflammatory activity by delaying the release and enhancing the oral bioavailability of PU. Moreover, pharmacokinetic studies carried out in rats highlighted the enhanced uptake efficiency of these PU-NPs in the blood. The rats administered with PU-NPs were found to have a higher PU level in the plasma compared to PU-administered controls. Thus, the results demonstrate the potential of PU-NPs over PU by overcoming the poor bioavailability of PU and accelerating its release in the blood. According to the results of the aforementioned in vitro and in vivo investigations, PU-NPs may be employed for treating inflammation.

Acknowledgments

The authors acknowledge National Institute of Pharmaceutical Education and Research (NIPER) and the Chandigarh Group of Colleges (CGC) Landran, Mohali, for providing all the necessary facilities to carry out the research work. The authors would also like to acknowledge their gratitude to Corbion for providing a gift sample of the Polymer PDLG 5002A.

Conflict of Interest

The authors declare no conflict of interest.

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