Evaluation of the Antioxidant, Anti-Inflammatory, and Antimicrobial Activity of a Cannabis sativa

Evaluation of the Antioxidant, Anti-Inflammatory, and Antimicrobial Activity of a Cannabis sativa-Infused African Product Used for Wound Healing

Source / Quelle: https://www.mdpi.com/1422-0067/27/15/6815

Abstract

Product Shezi (PS) is a polyherbal African traditional medicine (ATM) formulated from six known South African medicinal plants and is used for treating cutaneous wounds. However, its ethnopharmacological properties have not been scientifically validated. This study aimed to evaluate the antioxidant, anti-inflammatory, and antimicrobial activities of PS in vitro. Aqueous and methanolic extracts were prepared and qualitatively screened for phytochemical constituents. Cytotoxicity in fibroblasts and macrophages was assessed using an ATP-based viability assay. Antioxidant activity was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and a hydrogen peroxide (H2O2)-induced oxidative stress model. Anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated macrophages were measured using the Griess reagent system, a human prostaglandin E2 (PGE2) ELISA, and a bovine serum albumin (BSA) anti-denaturation assay. Antimicrobial activity was assessed by twofold serial broth microdilution, agar well diffusion, and a crystal violet biofilm assay. Phytochemical screening confirmed the presence of saponins, alkaloids, tannins, glycosides, terpenoids, flavonoids, and steroids. PS exhibited IC10 values of 3 and 10 μg/mL in fibroblasts and macrophages, respectively. PS (1–5 μg/mL) showed 70% DPPH scavenging potential and potent H2O2 cytoprotection (p < 0.001). Nitric oxide inhibition was non-significant (p > 0.05) whilst PGE2 decreased significantly (p < 0.05) compared to LPS-stimulated cells. BSA denaturation was inhibited in a dose-dependent manner (p < 0.05). Staphylococcus aureus and S. epidermidis were the only microorganisms susceptible to PS (MIC 650–5000 μg/mL) with low SI (SI ≈ 0.05–0.06), while others were resistant. PS showed no antibiofilm activity (crystal-violet assay). Overall, PS demonstrated antioxidant and anti-inflammatory activities and selective antibacterial effects against planktonic bacteria in vitro. However, further optimization of extraction methods, detailed mechanistic studies, and in vivo investigations are warranted to substantiate therapeutic relevance.

Keywords:

wound healing; African traditional medicine; anti-inflammation; antioxidant; antimicrobial; KwaZulu-Natal

1. Introduction

Wound healing is a complex reparative process consisting of four overlapping phases: hemostasis, inflammation, proliferation, and remodeling or maturation. The inflammatory phase represents an essential protective response to tissue injury and pathogen invasion. This process is crucial in achieving complete wound healing. The chief goal of this phase in the wound healing process is to purge cellular debris and phagocytose all the pathogens and foreign substances from the wound site [1]. However, the inflammatory phase is susceptible to interruption by both endogenous and exogenous factors, which may delay progression to the proliferative and remodeling stages [2]. The delay in wound healing affects the patient’s quality of life and healthcare systems. A recent study by Holzer-Geissler et al. [3] indicated that prolonged inflammation contributes to the development of chronic wounds. Dysregulated inflammation typically promotes membrane alterations, increases protein denaturation, and results in scar formation [4]. More broadly, chronic inflammation has been implicated in the pathogenesis of multiple disorders, including type 2 diabetes and aging-related conditions [5]. The inflammatory phase in chronic wounds, such as diabetic foot ulcers, lasts longer than that of acute wounds [6].

Precise regulation of inflammation and oxidative stress is a critical factor in complete wound healing. During the inflammatory phase, reactive oxygen species (ROS), such as hydrogen peroxide, hydroxyl radical, and nitric oxide, are produced by inflammatory cells to initiate the wound-healing process [7]. ROS are produced in low concentrations as a host response mechanism to eliminate infections at the wound site [8]. However, excessive ROS production leads to oxidative stress, disrupting membrane integrity. Disproportionate ROS production also impairs lipid and protein homeostasis and damages extracellular matrix components, thereby compromising healing [9]. In the human body, low levels of nitric oxide are essential for enhancing antimicrobial activity, especially after injury; thus, excessive concentrations contribute to prolonged inflammation [10]. Cytokines, as well as other mediators of inflammation such as prostaglandins, are also secreted during an inflammatory response [11]. An increase in pro-inflammatory cytokines, such as IL-1 and TNF-α, on the wound site promotes prostaglandin synthesis [12]. In inflamed tissue, prostaglandin E2 is released in excess, leading to peripheral nociceptor activation [13]. Overproduction of prostaglandins via cyclooxygenase pathways during wound healing results in pain. Therefore, persistent oxidative stress and unresolved inflammation are hallmarks of chronic, non-healing wounds [14]. In general, antioxidants are grouped into natural (enzymic antioxidants) and synthetic antioxidants. Synthetic antioxidant therapies have been developed to combat oxidative stress in cutaneous wounds. It has been reported that synthetic antioxidants are associated with some limitations, including low bioavailability and reduced bioactivity when topically applied directly to wounds [15].

Bacterial infection has been regarded as the major cause of delayed wound healing [16]. In the first week of injury to the dermal tissue, Gram-positive bacteria predominate the wound site. After a week, Gram-negative bacteria then colonize the wound [17]. Many pathogenic bacteria adhere to the wound surface and form biofilm-structured microbial communities embedded within a protective extracellular polymeric matrix. Species such as Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) are particularly adept at forming biofilms, which enhance their persistence and thus exacerbate infections and compromise wound healing [18]. These infections complicate wound management and further deteriorate clinical outcomes [19]. A recent study has shown a tight relationship between microbial infection and the wound-healing process [20]. Bacterial tolerance to antimicrobial agents at lethal concentrations has been attributed to their presence as a biofilm at the wound site [21]. Emerging evidence suggests that biofilm resistance to antimicrobial agents is 1000 times higher than that of planktonic cells [22]. This is largely due to the antibiotic penetration barrier, which consists of a specialized polysaccharide complex surrounding the biofilm bacteria [23]. Although antibiotic resistance may arise naturally, inappropriate and excessive antibiotic use has accelerated the emergence of multidrug-resistant (MDR) strains, contributing to treatment failure, increased healthcare costs, and heightened morbidity and mortality [24]. The widening gap between antimicrobial development and the rapid evolution of resistance underscores the urgency of identifying alternative therapeutic strategies.

Non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen, acetaminophen, and diclofenac are widely used in humans to inhibit inflammation [25]. The efficacy of NSAIDs in modulating inflammation is achieved by inhibiting prostaglandin synthesis and the cyclooxygenase enzyme [26]. Since inflammation is involved in many medical conditions, including wounds, the demand for anti-inflammatory drugs is very high. Antibiotics for wound-related infection may include vancomycin, ceftriaxone, gentamicin, and ciprofloxacin. One limitation of using conventional anti-inflammatory and antimicrobial drugs already on the market is the potential for severe complications and side effects [26]. Side effects may include gastrointestinal ulceration, stroke, hyperglycemia, perforation, and acute renal ulcer after oral administration [27]. Therefore, a search for alternative anti-inflammatory and antimicrobial agents from medicinal plants with reduced unwanted side effects associated with conventional treatments is warranted.

Medicinal plants have gained considerable attention in the field of wound healing in the past decade. Recent studies demonstrated that African traditional medicine (ATM) products possess antioxidant and anti-inflammatory activities [27]. These ATM products have been demonstrated to promote anti-inflammatory activities by inhibiting nitric oxide production, the lipoxygenase enzyme, and prostaglandin synthesis [28]. Their continued relevance is supported by data showing that over 80% of people on the African continent rely on traditional medicine as their primary source of healthcare. This includes treatment of skin-related diseases and associated infections. Globally, there has been increasing interest in studying medicinal plants as alternative treatments for inflammation to reduce limitations of conventional medicines [29]. However, there are limited ethnopharmacological studies on ATM products used in wound treatment. Among ATM products traditionally used for the treatment of wounds in South Africa is Product Shezi (PS), a Cannabis sativa (C. sativa)-infused polyherbal ATM formulated by a traditional healer in KwaZulu-Natal using five additional known medicinal plants for topical wounds. Product Shezi is a polyherbal African traditional medicine used for the topical treatment of wounds and is applied once daily according to the traditional healer’s instructions. It is formulated from six medicinal plants, with Cannabis sativa constituting 50% of the final preparation, while the remaining 50% consists of five known South African medicinal plants, including Drimia altissima (L.f.). Ker Gawl., Albuca fastigiata Dryand, Bulbine latifolia (L.f) Spreng, Hypoxis hemerocallidea Fisch., C.A. & Ave-Lall, and Hypericum aethiopicum Thumb. The interest in PS is based on anecdotal evidence from the traditional healer regarding its efficacy in treating chronic wounds (Supplementary Figure S1). Therefore, the aim of the study was to evaluate the in vitro antioxidant, anti-inflammatory, and antimicrobial activities of PS.

2. Results

2.1. Qualitative Phytochemical Analysis

The phytochemical analysis studies indicated the presence of all the prominent phytochemicals in PS (Table 1).

Table 1. Phytochemical parameters of Product Shezi.

2.2. Cytotoxicity Activity

A dose-dependent reduction in fibroblast and macrophage cell viability was observed following treatment with increasing concentrations of PS, compared to the untreated control. Higher concentrations of PS above 50 µg/mL induced the most pronounced cytotoxic effects in both fibroblast and macrophage cells (Figure 1). The IC50 of PS on fibroblast cells was determined to be 23.68 µg/mL (Figure 1), with an IC10 value of 3 µg/mL. For macrophage cells, the IC50 value was established at 32.47 µg/mL and the IC10 at 10 µg/mL (Figure 1B). Concentrations ranging from the IC10 and lower were used for further experiments.

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