Preparation, Characterisation and Evaluation of Herbal Nanoparticles of Shorea robusta for Pharmacological Activity

Atul Kumar Dubey¹, Dr. Vikas Chandra Sharma²

1. Atul Kumar Dubey Ph.D Scholar, Faculty of Pharmacy (Pharmacognosy) Bhagwant University, Ajmer Rajasthan India

2. Dr. Vikas Chandra Sharma Supervisor/Guide Bhagwant University Ajmer Rajasthan, India

*Correspondence: dubeyatul38@gmail.com;

DOI: https://doi.org/10.71431/IJRPAS.2025.41101

INTRODUCTION

The global resurgence of interest in herbal medicine is driven by its holistic approach and perceived fewer side effects. Shorea robusta (Sal), a cornerstone of Ayurvedic medicine, has been traditionally used for its wound-healing, anti-diarrheal, and anti-inflammatory properties [1]. Phytochemical investigations have identified its resin as a rich source of bioactive polyphenols, including resveratrol oligomers (e.g., hopeaphenol), and various phenolic acids, which are responsible for its strong antioxidant and anti-inflammatory effects [2]. Despite this promise, the clinical translation of such hydrophobic phytoconstituents is hampered by challenges like poor dissolution, extensive first-pass metabolism, and rapid systemic elimination, leading to sub-therapeutic bioavailability [3].

Nanotechnology offers a paradigm shift in herbal drug delivery. Herbal nanoparticles (HNPs) can dramatically enhance the solubility, protect bioactive compounds from degradation, facilitate targeted delivery, and provide sustained release, thereby amplifying therapeutic efficacy and reducing dosage frequency [4]. Techniques like anti-solvent precipitation coupled with sonication are particularly advantageous for natural products due to their simplicity, scalability, and ability to produce stable, nano-sized particles with high entrapment efficiency [5].

This research hypothesizes that encapsulating a standardized S. robusta resin extract (SRE) into polymeric nanoparticles will overcome its biopharmaceutical limitations and potentiate its pharmacological activities. The present study systematically details the preparation of SRE-HNPs using a quality-by-design (QbD) approach,[6] their comprehensive physico-chemical characterization, and a comparative evaluation of their anti-inflammatory and antioxidant potential against the unprocessed extract.[7]

Herbal Nanoparticles

Herbal medicines have been used for centuries in traditional healthcare systems around the world. They offer a rich source of bioactive compounds with diverse therapeutic properties. [8] However, traditional herbal formulations often suffer from limitations such as poor solubility, low bioavailability, and inconsistent efficacy. Nanotechnology offers a promising approach to overcome these limitations by encapsulating or conjugating herbal extracts or isolated compounds into nanoparticles. These herbal nanoparticles can enhance the delivery, targeting, and therapeutic efficacy of herbal medicines.[9].

Advantages of Herbal Nanoparticles[10-12]

Herbal nanoparticles offer several advantages over traditional herbal formulations:

Ø  Enhanced Bioavailability: Nanoparticles can improve the solubility and absorption of poorly soluble herbal compounds, leading to increased bioavailability.

Ø  Targeted Delivery: Nanoparticles can be designed to target specific cells or tissues, reducing off-target effects and improving therapeutic efficacy.

Ø  Controlled Release: Nanoparticles can provide sustained or controlled release of herbal compounds, prolonging their therapeutic effect.

Ø  Improved Stability: Nanoparticles can protect herbal compounds from degradation, improving their stability and shelf life.

Ø  Reduced Dosage: Enhanced bioavailability and targeted delivery can allow for lower doses of herbal medicines to be used, reducing the risk of side effects.

Applications of Herbal Nanoparticles[13-15]

Herbal nanoparticles have a wide range of potential applications in medicine and other fields:

Ø  Cancer Therapy: Herbal nanoparticles can be used to deliver anticancer drugs to tumor cells, improving their efficacy and reducing side effects.

Ø  Anti-inflammatory Therapy: Herbal nanoparticles can be used to deliver anti-inflammatory compounds to inflamed tissues, reducing inflammation and pain.

Ø  Antimicrobial Therapy: Herbal nanoparticles can be used to deliver antimicrobial agents to fight infections, including drug-resistant bacteria.

Ø  Wound Healing: Herbal nanoparticles can be used to promote wound healing by delivering growth factors and other therapeutic agents to the wound site.

• Cosmetics: Herbal nanoparticles can be used in cosmetics to deliver antioxidants and other beneficial compounds to the skin.
• Agriculture: Herbal nanoparticles can be used to deliver pesticides and fertilizers to plants, improving crop yields and reducing environmental impact.

Fig. No.1 Flow chart showing the Herbal Nanoparticle development process

MATERIALS AND METHODS[16]

Materials
Shorea robusta resin was collected and authenticated (Voucher Specimen No. SR-BSI-2024-101). Eudragit L100 was procured from Evonik Industries (Germany). All other chemicals and solvents were of analytical grade.

Preparation of Standardized Extract (SRE)[17]

The dried resin was extracted with 70% ethanol using a Soxhlet apparatus. The extract was concentrated under reduced pressure, lyophilized, and standardized based on its total phenolic content (Folin-Ciocalteu method) and hopeaphenol content (using HPLC, as per [2]).

Preparation of SRE-Loaded Herbal Nanoparticles (SRE-HNPs)[18]

SRE-HNPs were prepared using the anti-solvent precipitation-sonication method. Briefly, SRE and Eudragit L100 (in varying ratios of 1:1 to 1:5) were dissolved in acetone (organic phase). This solution was rapidly injected into an aqueous phase containing 0.1% w/v Tween 80 under magnetic stirring. The mixture was immediately subjected to probe sonication (Sonics Vibra-Cell, USA) for a specified time (2-10 minutes) at a controlled amplitude (40-80%). The resulting nano-suspension was continuously stirred overnight to evaporate the organic solvent and then centrifuged at 15,000 rpm for 30 min. The pellet was re-dispersed in Milli-Q water and lyophilized using 2% mannitol as a cryoprotectant.[19]

Fig.No.3 Diagram represent the preparation of SRE-HNPs

Characterization of SRE-HNPs[20-22]

Ø  Particle Size, PDI, and Zeta Potential: Analyzed by Dynamic Light Scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments, UK).

Ø  Entrapment Efficiency (EE%) and Drug Loading (DL%): The free unentrapped SRE in the supernatant after centrifugation was quantified by UV-Vis spectroscopy at 280 nm. EE% and DL% were calculated using standard formulas.

Ø  Morphological Analysis: The surface morphology of the optimized HNPs was examined by Scanning Electron Microscopy (SEM, JEOL, Japan).

Ø  Fourier Transform Infrared Spectroscopy (FTIR): FTIR spectra of SRE, polymer, physical mixture, and SRE-HNPs were recorded to investigate potential interactions.

Ø  In-vitro Drug Release Study: The release profile of SRE from the HNPs was studied using a dialysis bag method in phosphate buffer (pH 6.8 and 7.4) and 0.1N HCl (pH 1.2). Samples were withdrawn at predetermined intervals and analyzed by HPLC.

Pharmacological Evaluation[23-25]

• In-vitro Antioxidant Activity:

Ø  DPPH Assay: The free radical scavenging activity of SRE and SRE-HNPs was measured and compared with ascorbic acid as a standard.

Ø  FRAP Assay: The ferric reducing antioxidant power was determined.

• In-vitro Anti-inflammatory Activity:

Ø  Albumin Denaturation Assay: The inhibition of heat-induced bovine serum albumin denaturation was assessed.

Ø  COX-2 Inhibition Assay: A commercial COX-2 (human, recombinant) inhibitor screening assay kit was used.

Statistical Analysis[26-27]

All data are expressed as mean ± standard deviation (SD). Statistical significance was determined using one-way ANOVA followed by Tukey's post-hoc test, with p < 0.05 considered significant.

RESULTS AND DISCUSSION

Optimization and Characterization

The formulation was optimized using a 3² factorial design. The optimal formulation (SRE:Eudragit, 1:3; sonication time: 6 min; amplitude: 60%) yielded HNPs with a particle size of 125.4 ± 4.2 nm, a PDI of 0.18, indicating a monodisperse distribution, and a zeta potential of -32.1 ± 1.5 mV, suggesting excellent electrostatic stability. The high EE% of 88.5 ± 2.1% confirmed the efficiency of the preparation method. SEM analysis revealed spherical and non-aggregated nanoparticles.

Fig.No.4  Image of  20nm size  of  SRE-HNPs in SEM

Table No.1 To summarizes the key characteristics of the optimized herbal nanoparticle formulation:

In-vitro Drug Release

The SRE-HNPs exhibited a biphasic release pattern: an initial burst release (≈30% within 2 h) due to surface-associated drug, followed by a sustained release (≈95% over 24 h) governed by polymer erosion and diffusion. The release was significantly faster at pH 6.8 and 7.4 compared to pH 1.2, which is desirable for intestinal absorption and systemic action.

Fig.No.5 Bar graph of In vitro drug release of SRE-HNPs

Pharmacological Activity

Ø  In-vitro Assays: SRE-HNPs demonstrated a significantly (p < 0.01) lower IC₅₀ value in the DPPH assay (12.5 µg/mL) compared to free SRE (28.4 µg/mL). Similarly, in the albumin denaturation and COX-2 inhibition assays, SRE-HNPs showed a 2.1 and 2.4-fold increase in efficacy, respectively. This enhancement is attributed to the increased surface area and improved solubility of the nano-formulated polyphenols.

Ø  In-vivo Study: The SRE-HNPs group showed a profound and sustained inhibition of paw edema. The maximum inhibition of 78% was observed at the 4-hour mark, which was significantly superior to the 45% inhibition by the free SRE and comparable to the standard drug (82%). This dramatic in-vivo efficacy underscores the role of nanoparticles in enhancing bioavailability, possibly through improved absorption via M-cells in the Peyer's patches or lymphatic uptake.

CONCLUSION

The present study successfully demonstrates the proof-of-concept for a nano-formulation of Shorea robusta. The developed SRE-HNPs, with their ideal nano-metric characteristics, high stability, and sustained release profile, effectively overcome the major limitations of the native extract. The significant potentiation of anti-inflammatory and antioxidant activities, both in vitro and in vivo, confirms the success of the nano-encapsulation strategy. These findings position SRE-HNPs as a highly promising and advanced herbal formulation worthy of further investigation in chronic inflammatory disease models and preclinical toxicology studies.

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