Twin-screw melt granulation of Punica granatum leaves enhances in vitro ellagitannin stability and enables solid dosage form development

Abstract

Ellagitannins (ETs) from Punica granatum leaves are susceptible to degradation under less acidic pH conditions, which may compromise their use as active ingredients in oral solid dosage forms. This study investigated lipid-based twin-screw melt granulation (TSMG) as a downstream strategy to improve ET stability during an in vitro pH-stress assay at pH 6.8 and facilitate solid dosage form development. Two glyceryl behenate-based binders were initially screened, followed by systematic evaluation of screw configuration, feed rate, screw speed, and filler grade. Granatin B stability was monitored by HPLC-DAD over 6 h, and the resulting granules were characterized regarding particle size, morphology, flowability, and downstream processability. Compritol® 888 ATO provided the lowest apparent Granatin B degradation constant (0.194 ± 0.009 h⁻¹), compared with Compritol® HD5 ATO granules (0.281 ± 0.004 h⁻¹) and the ungranulated spray-dried extract (0.374 ± 0.010 h⁻¹). Subsequent process optimization shifted the particle-size distribution toward larger particle classes and improved flow properties. The optimized granules were incorporated at 50% (w/w) into mechanically resistant tablets that disintegrated within 20–30 min and enabled ET release despite the high lipid-matrix content, indicating that the matrix did not impose a markedly prolonged-release profile. Incorporation into hydroxypropyl methylcellulose acetate succinate (HPMC-AS) capsules was also feasible, although ET recovery depended on the proportion of extragranular hydrophilic excipients. Overall, lipid-based TSMG provided an in vitro proof of concept for integrating improved ET stability under pH-stress conditions with granule manufacturability and solid dosage form development. Further studies using sequential biorelevant media and appropriate in vivo models are required to establish gastrointestinal preservation and site-specific intestinal delivery.

Introduction

Ellagitannins (ETs) are a large group of bioactive compounds belonging to the hydrolysable tannin class [1]. The consumption of ETs has been associated with health-promoting effects in several pathologies, owing to their broad biological activities, including anti-inflammatory, anticancer, antiviral, and antimicrobial properties. Furthermore, ETs exert beneficial effects on gut health by modulating the intestinal microbiota, stimulating the production of short-chain fatty acids, and enhancing immune function [[2], [3], [4], [5], [6], [7]].

These biological activities are particularly relevant to inflammatory bowel diseases, such as ulcerative colitis, which are characterized by persistent mucosal inflammation, oxidative stress, and impaired intestinal barrier function [8,9]. Preclinical studies have shown that punicalagin and pomegranate polyphenolic preparations containing ETs can attenuate experimental colitis by strengthening the intestinal epithelial barrier, reducing oxidative stress, and modulating inflammatory mediators and signaling pathways, including AMPK/NF-κB/STAT3, TNF-α, IL-1β, COX-2, iNOS, and miR-145/p70S6K1/HIF-1α [10,11]. Preliminary clinical evidence indicated a higher clinical response after four weeks of adjunctive treatment with pomegranate peel extract, containing ETs, than with placebo in patients with ulcerative colitis. However, the between-group difference did not reach statistical significance, indicating that adequately powered clinical trials are still required to confirm its therapeutic efficacy [12].

Despite their promising bioactivities, ETs exhibit low oral bioavailability due to their large molecular weight, high polarity, and vulnerability to degradation by gastrointestinal pH, enzymes, and gut microbiota, which limits their systemic absorption [13,14]. Instead, their microbial derived metabolites, urolithins, are more readily absorbed and have been implicated in antioxidant, anti-inflammatory, neuroprotective, and chemopreventive effects [1,3,15,16]. The conversion of ETs into urolithins by gut microbiota begins in the distal gastrointestinal tract, where ETs release hexahydroxydiphenoyl (HHDP) or dehydrohexahydroxydiphenoyl (DHHDP) units that spontaneously lactonize into ellagic acid. This intermediate is subsequently metabolized into urolithins through a series of microbial transformations [13,14]. However, the instability of ETs at less acidic pH conditions often leads to premature oxidation of HHDP and DHHDP groups, impairing ellagic acid release and urolithin formation, thus compromising their therapeutic potential [13,14,[16], [17], [18], [19], [20], [21]]. Therefore, the development of a formulation capable of protecting ETs from premature degradation under less acidic to neutral gastrointestinal conditions (pH 6.8) is critical to preserving their integrity and ensuring effective microbial conversion in the colon [8].

To overcome this limitation, ETs must be protected from premature degradation during gastrointestinal transit while remaining available for release in the distal intestine, where microbial conversion occurs. An appropriately designed excipient-based matrix may reduce the contact of ETs with the gastrointestinal medium and delay their oxidation without preventing subsequent release. In this context, TSMG represents a suitable solvent-free strategy for producing granules capable of modulating ET stability and release.

TSMG is a continuous manufacturing technique that uses heat and mechanical shear to agglomerate powders in the presence of a meltable binder. Compared with conventional wet granulation, TSMG eliminates the addition of granulation solvents and the subsequent drying step, shortens processing time, and enables greater control over critical process variables [22]. Previous studies have primarily applied TSMG to conventional active pharmaceutical ingredients to improve powder flow, tabletability, compressibility, taste masking, solubility, and drug release [20,[23], [24], [25], [26]]. TSMG may also promote matrix formation or partial particle coating by the molten binder, thereby limiting exposure to moisture or unfavorable pH conditions and improving the stability of sensitive compounds [22,27]. Binder selection in TSMG must consider both the thermal sensitivity of the active material and the intended function of the resulting matrix. Hydrophilic amorphous polymers, including hydroxypropyl cellulose, copovidone, and povidone, can improve granule formation and matrix stability when their glass-transition behavior, viscosity, and compatibility with the active material are suitable. HPC EF, Kollidon® VA64, and Kollidon® 12 PF, for example, have been used to improve binder distribution and preserve the integrity of lipid-based sustained-release matrices produced by TSMG [28]. However, processing with amorphous polymeric binders depends on formulation-specific softening and plasticization behavior and may require relatively high temperatures [29]. This aspect is particularly relevant to herbal extracts, which contain chemically diverse constituents with potentially different and often insufficiently characterized thermal stabilities. Moreover, possible physical and chemical interactions between multiple extract constituents and polymeric carriers may complicate the prediction of processability and stability [30].

Lipid binders may offer an alternative for thermally sensitive, chemically complex materials because they generally melt within a comparatively moderate and well-defined temperature range while forming matrices capable of controlling medium penetration and compound release. Glyceryl dibehenate (Compritol® 888 ATO), which melts at approximately 65–77°C, is widely used as a hydrophobic matrix former, modified-release agent, and protective coating material. These properties may reduce contact between ETs and less acidic gastrointestinal media, thereby limiting premature degradation while allowing subsequent intestinal release [25,31,32].

Although TSMG has been widely investigated using conventional active pharmaceutical ingredients, its application to herbal extracts remains limited. To the best of our knowledge, only one previous study from our research group applied TSMG to a spray-dried herbal extract. In that study, glyceryl behenate improved the flowability and tabletability of a Spondias mombin leaf extract and enabled modulation of ellagitannin release. The release results also suggested a potential protective effect against tannin degradation [33]. However, this interpretation remained preliminary because tannin stability and the underlying protection mechanism were not systematically investigated.

Spray drying remains one of the most commonly employed approaches for stabilizing bioactive constituents in herbal extracts. The incorporation of extracts into matrices composed of maltodextrin, gum arabic, proteins, or other carrier materials can improve bioactive retention and storage stability while enabling their conversion into dry powders [[34], [35], [36]]. Nevertheless, the protective performance of spray-dried systems depends strongly on carrier composition and drying conditions, and the resulting powders may still require downstream processing to achieve adequate flowability, tabletability, and release properties [30]. Thus, spray drying alone does not necessarily address all the challenges associated with the stabilization of dried herbal extracts and their subsequent processing into tablets, particularly protection against pH-dependent degradation during gastrointestinal transit. Therefore, further investigation of lipid-based TSMG is warranted to determine whether the preliminary protective effect observed for hydrolysable tannins can be systematically applied to improve ET stability and support solid dosage form development.

Among the natural sources of ETs, Punica granatum (pomegranate) stands out due to the remarkable diversity and abundance of these compounds, especially granatin B [37]. In particular, pomegranate leaves have garnered increasing attention due to their therapeutic potential and high content of ETs [38,39]. Moreover, these leaves represent a sustainable raw material, as they are commonly regarded as agricultural and industrial by-products from pomegranate cultivation and processing [40,41]. Their valorization aligns with current strategies for circular bioeconomy and the development of phytopharmaceuticals targeting the gut microbiota, inflammation, and immune regulation [20,40,42,43].

Based on this rationale, we hypothesized that rational lipid-binder selection combined with systematic evaluation of TSMG process parameters could attenuate ET degradation under pH conditions known to compromise their intact structures and facilitate the use of spray-dried P. granatum leaf extract as an active ingredient in solid dosage forms. Therefore, this study aimed to investigate whether lipid-based TSMG could improve ET stability during an in vitro pH-stress assay at pH 6.8 while producing granules with suitable technological properties for tablet and capsule development. This granule-engineering framework explores TSMG as an alternative downstream processing strategy for herbal extracts by integrating improved pH stability, manufacturability, and feasibility for incorporation into solid dosage forms.

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Materials

Ethanol (Dinâmica, Brazil), Lactose (Flowlac 100®, Meggle pharma, Germany), Xanthan gum (Sigma-Aldrich®, Germany), Aerosil® (Evonik, Germany), Behenoyl polyoxyl-8 glycerides – Compritol® HD5 ATO (CPT-HD5) (Gattefossé, France), Glyceryl dibehenate – Compritol® 888 ATO (CPT 888) (Gattefossé, France), Microcrystalline cellulose 101 (MCC 101) (Vivapur®, JRS Pharma, Germany), Microcrystalline cellulose 200 (MCC 200) (Vivapu®, JRS, Germany), Dimethyl sulfoxide (DMSO) (Sigma®, Germany), Phosphate disodium anhydrous kindly donated by Chemische Fabrik Budenheim KG (Germany), Citric acid anhydrous (Emprove®, Merck, Germany), PVDF 0.45 μm syringe filters (Thermo Scientific®, Germany), Syringe filters 0.22 μm (Chromafil® RC-20/15 MS, Germany), Methanol HPLC grade (Lichrosolv®, Supelco, Germany), Trifluoroacetic acid (TFA) 99% (Thermo Scientific®, Germany), Ellagic acid standard 98% (Thermo Scientific®, Germany), Formic acid 99% (Sigma-Aldrich®, Germany), C18 reversed phase column (HS, particle size 5 μm, 25 cm × 4.6 mm, Supelco®, Germany), Safeguard C18 (Phenomenex®, 4 × 3 mm, USA). Tricalcium citrate four hydrate (TCC TB) (Jungbunzlauer, Germany), Croscarmellose sodium (CCS) (Vivasol®, JRS Pharma GmbH & Co. KG, Germany), Sodium starch glycolate (SSG) (Glycolys®, Roquette, Germany), Hydroxypropylmethylcellulose acetate succinate capsule (HPMC-AS) (Lonza, USA).

Janaina Carla Barbosa Machado, Joyce Cristina da Silva, Camylla Janiele Lucas Tenório, Gabriel Victor Batista Leite, Magda Rhayanny Assunção Ferreira, Kristina Steffens, Karl Gerhard Wagner, Luiz Alberto Lira Soares, Twin-screw melt granulation of Punica granatum leaves enhances in vitro ellagitannin stability and enables solid dosage form development, Journal of Drug Delivery Science and Technology, Volume 127, Part 1, 2027, 108882, ISSN 1773-2247, https://doi.org/10.1016/j.jddst.2026.108882.