Preparation and Investigation of Artemisia annua L.-Loaded Alginate Hydrogels with Excipients

Abstract

Background: Artemisia annua L. is a medicinal plant with documented antimicrobial, antioxidant, and anti-inflammatory properties. Although widely studied for internal therapeutic applications, its topical use—especially in hydrogel-based systems—has not been thoroughly investigated. The aim of this study was to develop sodium alginate hydrogels containing Artemisia annua extract, supplemented with hyaluronic acid and dexpanthenol, and to evaluate their physicochemical characteristics as well as their biological activities in vitro and in vivo.

Methods: Select bioactive constituents of the Artemisia annua extract were quantified using liquid chromatography coupled with electrospray ionization mass spectrometry (LC-ESI-MS). Hydrogels were prepared by cross-linking sodium alginate with a calcium carbonate–glucono-delta-lactone system and were formulated with or without hyaluronic acid and dexpanthenol. Physicochemical evaluations included measurements of moisture content, water-retention capacity, gelation time, and pH. The hydrogel microstructure was examined by scanning electron microscopy (SEM). Antioxidant activity was assessed using three methods: the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, the ferric reducing antioxidant power (FRAP) assay, and the cupric reducing antioxidant capacity (CUPRAC) assay. Biocompatibility and regenerative effects were analyzed using cell viability assays and an in vitro scratch wound model on human keratinocyte cells. In vivo wound-healing efficacy was examined in rats with full-thickness skin excisions.

Results: The extract contained high levels of methylated flavonoids and sesquiterpenes characteristic of Artemisia annua. Hydrogels supplemented with hyaluronic acid and dexpanthenol exhibited improved hydration stability and higher porosity. All formulations demonstrated measurable antioxidant activity, and those containing hyaluronic acid showed the strongest effects. The preparations were biocompatible and enhanced keratinocyte migration in vitro, with the combined hyaluronic acid–dexpanthenol formulation promoting the fastest wound closure. In vivo, Artemisia annua hydrogels accelerated wound healing by two to three days compared with untreated wounds.

Conclusions: These results confirm the promise of Artemisia annua hydrogels for topical wound care and highlight the beneficial contributions of hyaluronic acid and dexpanthenol to their structural and therapeutic performance.

Introduction

Artemisia annua L. (AA) is a medicinal plant that belongs to the Asteraceae family, commonly known as sweet wormwood or Qinghao [1]. It is an annual herb native to Asia, now distributed in Europe, Africa, and North America. It has been used in traditional Chinese medicine for thousands of years for the treatment of fever and infectious diseases such as malaria [2]. The medicinal value of AA extends beyond malaria, as it has been explored for its antiviral, antifungal, antimicrobial, anticancer, and anti-inflammatory properties [1,3]. AA has shown potential antidiabetic effects by improving glucose metabolism and enhancing insulin sensitivity by reducing the glucose levels and increasing insulin levels in the sera of diabetic rats [4,5]. A wide range of phytochemicals, including sesquiterpenoids, flavonoids, coumarins, lipids, phenolics, purines, steroids, triterpenoids, aliphatic substances, and the primary bioactive compound artemisinin, have been described from AA [6,7].

There is an extensive body of evidence available on the internal use of the herb; however, there are few studies on the topical use of AA and its potential excipients. Huang et al. demonstrated the positive effect of the drug on atopic dermatitis in mice with a topical formulation of AA essential oil [8]. Bao et al. investigated the efficacy of artemisinin-loaded hydrogels in rats to promote wound healing and the anti-tumor effects of the formulations [9].

Hydrogels are three-dimensional, hydrophilic polymer networks that can retain large amounts of water, making them highly useful for biomedical applications such as wound healing, drug delivery, and tissue engineering [10,11]. Their biocompatibility, ability to mimic natural tissue environments, and capacity to control the release of therapeutic agents contribute to their widespread use in the medical and pharmaceutical fields.

Sodium alginate, a naturally derived polysaccharide from brown seaweeds, is widely used in the food, pharmaceutical, and biomedical industries for its biocompatibility and non-toxic properties [12,13]. It is characterized by its ability to form gels in the presence of divalent cations like calcium, which cross-link its polymer chains to create a stable gel structure [14]. In hydrogels, sodium alginate plays a key role by providing a structure for controlled drug release, cell encapsulation, and wound healing, due to its ability to absorb water and maintain a moist environment [15,16].

Hydrogels are considered highly effective for topical drug delivery due to their high water content, biocompatibility, and versatility in encapsulating various drug types [11]. Recent studies also highlight that hydrogel formulations can be engineered with specific polymer networks and active extracts to enhance antioxidant, antibacterial, and cell-proliferative properties, further supporting their therapeutic potential in skin applications like wound healing [17,18]. Their water-rich structure enables them to maintain a moist environment on the skin, which enhances drug absorption and aids in wound healing [19,20]. Hydrogels can also be modified to control drug release rates, allowing for sustained and localized drug delivery over extended periods [21]. Additionally, their three-dimensional polymeric network provides structural stability while facilitating efficient interaction between the drug and the skin surface [22].

Hyaluronic acid (HA), often used in topical formulations at concentrations ranging from 0.1% to 2%, provides powerful hydration by binding moisture to the skin, enhancing skin elasticity, and reducing the appearance of wrinkles [23]. In addition to its hydrating properties, HA also acts as a skin barrier enhancer, decreasing transepidermal water loss (TEWL) and protecting against environmental stressors [24].

Dexpanthenol (DP), a derivative of pantothenic acid (vitamin B5), is a biologically active compound that penetrates the skin efficiently, where it is converted to pantothenic acid, promoting wound healing, skin hydration, and cellular regeneration [25]. The concentration of dexpanthenol in topical formulations typically ranges from 2% to 5%, providing optimal skin penetration and enhancing wound healing and hydration effects. Proksch et al. showed that dexpanthenol-containing formulations can accelerate wound healing and reduce the time to wound closure, which is crucial for minimizing transepidermal water loss (TEWL) and lowering the risk of infection [26].

Transdermal drug delivery is a critical advancement in pharmaceutical science, enabling the controlled and sustained release of therapeutics through the skin directly into systemic circulation, thereby enhancing bioavailability, minimizing first-pass metabolism, and reducing adverse effects [27]. Matrix systems embedded in natural polymers offer biocompatible, biodegradable, and sustained drug release while minimizing skin irritation, simplifying application, and enhancing tissue integration—making them ideal for safe and effective drug delivery [28].

The aim of this study was to formulate a stable and well-tolerated drug carrier for AA extracts with different excipients, and to evaluate the anti-inflammatory, antioxidant, and wound-healing effects of these formulations. Hydrogels are potentially good drug carriers for external drug delivery, and the safety of their internal use of sodium alginate has been mentioned in a previous article [29]. In order to characterize the formulations, different dosage form studies were performed, and the moisture content, water retention, pH, and gelation time of the hydrogels were measured. The quantification of select bioactive constituents of AA was performed using LC-ESI-MS. In vitro biocompatibility, cytotoxicity, wound-healing, and anti-inflammatory activity assays were performed on human keratinocyte cells (HaCaT). DPPH, FRAP, and CUPRAC antioxidant assays were performed to investigate the antioxidant effects of the formulations. The in vivo anti-inflammatory effects of the formulations were investigated in a rat paw edema model. The rats were incised and monitored continuously for two weeks to investigate the wound healing-promoting effects of the selected preparations.

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Materials

A commercial dried extract of Artemisia annua L. (drug-to-extract ratio 1:30), manufactured using a water/ethanol solvent system, was obtained from Natürlich lang leben UG© (Coburg, Germany; batch number: 73162). According to the certificate of analysis provided by the manufacturer, the extract was produced using a water/ethanol solvent system and subsequently dried to obtain the final powder. Low-viscosity-grade sodium alginate was obtained from BÜCHI Labortechnik AG (Flawil, Switzerland). D-(+)-glucono-δ-lactone (CAS No.: 90-80-2), D-Panthenol (CAS No.: 81-13-0), hyaluronic acid sodium salt from Streptococcus equi (CAS No.: 9067-32-7), calcium carbonate (CAS No.: 471-34-1), and FRAP assay kits (P. No.:MAK509) were purchased from Sigma-Aldrich (St. Louis, MI, USA). The MTT dye 2-4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide, Dulbecco’s Modified Eagle’s Medium (DMEM), phosphate-buffered saline (PBS), trypsin from porcine pancreas, ethylene-diamine-tetra-acetic acid (EDTA), heat-inactivated fetal bovine serum (FBS), L-glutamine, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 96% V/V% ethanol and (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) (CAS Number: 53188-07-1), L-ascorbic acid (CAS No.: 50-81-7), neocuproine (CAS No.: 484-11-7), copper (II) chloride dihydrate (CAS No.: 10125-13-0), and ammonium acetate (CAS No.: 631-61-8) were purchased from Sigma-Aldrich (Budapest, Hungary). Non-essential amino acid solution and penicillin–streptomycin mix, 12-well plates, 24-well plates, 96-well plates, and cell culture flasks were obtained from Thermo-Fisher (Darmstadt, Germany, CAS number: 156499). HaCaT cell lines (human keratinocyte cells) were obtained from Cell Lines Service (CLS, Heidelberg, Germany).

Papp, B.; Szűcs, Z.; Gonda, S.; Cziáky, Z.; Kajtár, R.; Lekli, I.; Haimhoffer, Á.; Klusóczki, Á.; Józsa, L.; Pető, Á.; et al. Preparation and Investigation of Artemisia annua L.-Loaded Alginate Hydrogels with Excipients. Pharmaceuticals 2026, 19, 424. https://doi.org/10.3390/ph19030424

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