Assessing radical scavenging capacity of Sempervivum tectorum L. leaf extracts: An integrated high-performance thin-layer chromatography/in silico/chemometrics approach

The use of medicinal plants for the prevention and treatment of various diseases has been a longstanding practice in traditional medicine. Many herbs have been investigated for their ability to scavenge free radicals, which play a role in the development of various health disorders such as Alzheimer's disease, Parkinson's disease, and atherosclerosis. The presence of phytochemicals such as phenolic acids and flavonoids, and their glycosides, are often positively correlated with the radical scavenging properties of natural products.

One such medicinal plant is houseleek (Sempervivum tectorum L.), an evergreen plant with succulent leaves arranged in perennial basal rosettes [1]. In traditional medicine, fresh or dried leaves or juice squeezed from the leaves are used externally or internally to treat a wide range of ailments [2]. Squeezed juice from the leaves is applied to the skin for treating wounds, ulcers, burns, and warts, or applied directly into the ear to reduce inflammation [3]. Houseleek fresh leaves are used internally to treat slow digestion, high blood sugar level, nervousness and anxiety by children, insomnia, epilepsy, stomach ulcers, menstruation ailments, and throat and bladder inflammation [4], [5], [6]. Regardless of wide application in folk medicine, S. tectorum does not have a monograph in the European Pharmacopoeia [7]. The leaves of houseleek have been found to contain a variety of phenolic compounds, such as kaempferol, quercetin and their glycosides, as well as phenolic acids such as gallic and caffeic acids [8], [9], [10]. The radical scavenging capacity of houseleek extracts was determined using spectrophotometric methods, such as using DPPH and ABTS assays [10].

Spectrophotometric and chromatographic methods are widely used to assess the antioxidant capacity (AOC) of plant extracts. These methods can include techniques such as DPPH, ABTS, FRAP, CUPRAC, and various radical scavenging assays [11,12]. Electrochemical techniques are also gaining popularity as they are convenient and sensitive alternatives [13]. While these techniques provide information on the overall radical scavenging capacity of the extract, they do not provide information on the contributions of individual compounds to the activity. On the other hand, High-Performance Thin-Layer Chromatography (HPTLC) allows for the separation and visualization of individual radical scavengers within the extract, providing a more detailed understanding of their contribution to the overall activity. In addition, chromatography is the rapid, simple, and inexpensive method that does not require large quantities of samples and chemicals, and has the possibility of simultaneous analysis of a large number of samples under the same chromatographic conditions. Furthermore, the instrumentation used in these assays has led to greater accuracy and reproducibility. The results presented in the manuscript confirmed the feasibility of comparing all five HPTLC-RSC assays, two of which (HPTLC-TRP and HPTLC-TAC) are entirely optimized and developed as new assays.

Previously, Folin-Ciocalteu (FC), DPPH, and ABTS assays have been utilized for the assessment of AOC on the plate, with DPPH assay being the most commonly used method [14], [15], [16]. In this study, we introduce the use of HPTLC in combination with potassium hexacyanoferrate(III) total reducing power assay (TRP) and phosphomolybdenum total antioxidant capacity (TAC) assay. Table 1 provides an overview of the strengths, limitations, and molecular mechanisms of these antioxidant assays [17,18].

Computational chemistry methods are a valuable tool for evaluating the radical scavenging capacity (RSC) of various natural and synthetic antioxidants [19]. By examining the bond dissociation enthalpies (BDE) associated with hydrogen atom transfer (HAT) and ionization potentials (IP) related to single electron transfer (SET) mechanism, these methods can provide insight into the thermodynamic aspects of the antioxidant capacity (AOC) of individual constituents within the extract.

In this study, RSC of twenty houseleek leaf extracts in 70% ethanol solution was analyzed using five HPTLC-RSC assays. The HPTLC-TRP and HPTLC-TAC assays were developed and optimized for the first time. Principal component analysis (PCA) was used to group houseleek samples based on the AOC of individual zones and to identify the most significant radical scavengers responsible for classification between samples. Additionally, PCA was applied to find similarities between the five HPTLC-RSC assays and to propose a combination of two assays that is the most suitable for the detection of HAT-based and SET-based antioxidants from natural products. The BDE and IP values for the five identified antioxidants were calculated using DFT with M06–2X functional and 6–31G+(d,p) basis set in simulated aqueous and lipid environments to provide insights into their corresponding mechanisms of action.

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