Abstract: The Salvia L. genus (Lamiaceae) is largely used in the pharmaceutical and food industry. Several species of biological relevance are extensively employed in traditional medicine, including Salvia aurea L. (syn. S. africana-lutea L.), which is used as a traditional skin disinfectant and in wounds as a healing remedy; nevertheless, these properties have not been validated yet. The present study aims to characterize S. aureus essential oil (EO), unveiling its chemical composition and validating its biological properties. The EO was obtained by hydrodistillation and subsequently analyzed by GC-FID and GC-MS. Different biological activities were assessed: the antifungal effect on dermatophytes and yeasts and the anti-inflammatory potential by evaluating nitric oxide (NO)  production and COX-2 and iNOS protein levels. Wound-healing properties were assessed using the scratch-healing test, and the anti-aging capacity was estimated through the senescence-associated beta-galactosidase activity. S. aurea EO is mainly characterized by 1,8-cineole (16.7%), β-pinene (11.9%), cis-thujone (10.5%), camphor (9.5%), and (E)-caryophyllene (9.3%). The results showed an effective inhibition of the growth of dermatophytes. Furthermore, it significantly reduced protein levels of iNOS/COX-2 and simultaneously NO release. Additionally, the EO exhibited anti-senescence potential and enhanced wound healing. Overall, this study highlights the remarkable pharmacological properties of Salvia aurea EO, which should be further explored to develop innovative,  sustainable, and environmentally friendly skin products.

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1. Introduction

Medicinal plants have several organoleptic characteristics that contribute to their high value in the pharmaceutical, nutraceutical, cosmetic, and food industries. Their secondary metabolites are emerging as potential candidates for new anti-inflammatory and anti-fungal drugs [1–3]. Among others, aromatic species from the Lamiaceae family are well known for their richness in essential oils, demonstrating a wide range of biological effects, including, in particular, anti-inflammatory and antifungal [4–8]. One of the largest and most important genera of this family is the genus Salvia L., which comprises nearly 1000 species [9]. Some of these are cultivated worldwide for their culinary use as well as for medicinal purposes; in fact, they have long been employed in folk medicine [10,11]. Many species show important biological properties, including antibacterial, antifungal, antioxidant, anti-inflammatory,  anticancer, hypoglycemic, and antidementia effects [12,13].

Although this genus has been extensively studied, there is still a great interest in those species that can be cultivated (domesticated) and used for health-promoting properties. It is well known that secondary metabolites are the result of plant/environmental interactions. Therefore, studies on the domesticated population can provide relevant information on whether secondary metabolites and their related biological properties vary from the native population. Among these, Salvia aurea L. (syn. S. africana-lutea L.) is an important aromatic species that is used for medicinal purposes in the Western Cape (South Africa) and is cultivated in many parts of the world as an aromatic/medicinal plant as well as ornamental (urban furniture). This species is a grey-green branched aromatic shrub that grows to about 2 m, with petiolate leaves that accumulate its essential oil in glandular trichomes and presents characteristically large and golden-brown flowers [14]. It is native to South Africa, where its geographical distribution extends westwards from the Cape Peninsula towards Namaqualand as well as eastwards from the Cape in the direction of Port Alfred in the Eastern Cape Province. It is often called either “beach sage, sandsalie”,  which is typical of the dune environment, or “brown dune sage” for the characteristic color of its flowers [15,16]. 

S. aurea is known for its several uses in traditional medicine, notably as an antiinflammatory and medicinal wash for the treatment of wounds and skin affections. Furthermore, this species is also used for the healing of bronchitis and other respiratory ailments reinforcing its antimicrobial properties [17,18].

Dermatophytes, particularly those of Trichophyton, Epidermophyton, and Microsporum,  are frequently associated with superficial mycoses [19]; however, immunocompromised individuals can be affected by invasive infections [20].

During these infections, fungal epitopes [21,22] bind to Toll-like receptors (TLRs),  triggering the activation of a pro-inflammatory state. One of the most significant pathways associated with this condition is the nuclear factor kappa B (NF-κB) pathway, which involves the secretion of inflammatory cytokines and the synthesis of pro-inflammatory enzymes, for example, the inducible Nitric Oxide Synthase (iNOS) and the Cycloxygenase- 2 (COX-2) [23]. These enzymes are responsible for nitric oxide (NO) and prostanoid production, respectively [24,25].

Furthermore, dermatophytes often lead to skin lesions [26]. Therefore, wound healing is needed to regenerate the damaged tissue; otherwise, the wound becomes chronic [27]. 

Fungal infections are still an unmet clinical need since the etiological agents are often associated with resistance to therapy and lead to relapses, while conventional drugs are widely associated with adverse effects [28]. Moreover, several fungal strains are known to be resistant to current antifungal agents [29,30]. Anti-inflammatory drugs have also been linked to several adverse effects [31]. Therefore, it is crucial to develop new strategies that,  in addition to treating fungal infections, can also inhibit the pro-inflammatory state that is evoked during infection.

Given its traditional uses, it is likely that S. aurea might have concurrent antifungal and anti-inflammatory activities. However, these properties have been tested by a limited number of studies on a narrow range of microorganisms [32–34]. Furthermore, to the best of our knowledge, no studies have reported the wound-healing properties of S. aureus so far. Therefore, this work aims to characterize the essential oil of S. aureus (sandsalie) when domesticated in a coastal area of the island of Sardinia. Considering the oil variation in season and locality, this territory was chosen for its similarities with the South African Fynbos ecoregion, S. aurea native territory [35,36]. In addition, we validated some of the traditional uses ascribed to this species, particularly those related to antimicrobial,  anti-inflammatory, and wound healing uses. Anti-senescence effects were also disclosed to further promote interest in this plant.

2. Results

2.1. Chemical Composition of S. aurea Essential Oil 

S. aurea leaves were subjected to hydrodistillation, obtaining the essential oil (EO) with 0.54% (w/w) yield. In Table 1, EO-identified compounds and their relative percentages are listed by their elution order. Thirty-six compounds were identified, for a total of 98.8%.

Furthermore, 47.4% of the compounds are oxygenated monoterpenes, 28.8% hydrocarbon monoterpenes, 19.8% hydrocarbon sesquiterpenes, and 2.8% oxygenated sesquiterpenes.

Chemical analysis revealed that S. aurea oil’s most representative components were 1,8  cineole (16.7%), β-pinene (11.9%), cis-thujone (10.5%), camphor (9.5%), (E)-caryophyllene (9.3%), trans-thujone (6.9%), α-pinene (4.7%), camphene (3.9%), and α-humulene (3.0%).

2.2. Antifungal Effect of Salvia aurea

The microdilution broth method was used to assess the antifungal activity, as reported in Table 2. Trichophyton mentagrophytes, T. rubrum and Epidermophyton floccosum are the most sensitive strains to EO activity. Specifically, EO showed a fungicidal effect for T. mentagrophytes and E. floccosum.

2.3. Anti-Inflflammatory Potential of S. aurea

To assess the anti-inflammatory potential of S. aurea, we resorted to the lipopolysaccharide (LPS)-stimulated macrophages model. As expected, in the presence of LPS, the macrophages released NO to the medium, where it was detected as a nitrate ([NO] = 43.03 ± 7.74 µM). Interestingly, S. aurea EO reduced the NO release evoked by LPS dose-dependently (Figure 1A, IC50 = 1.07 µL/mL). The dose of 1.25 µL/mL was selected to unveil the underlying mechanisms as it inhibited NO production by more than 50%, was devoid of toxicity (Figure 1B),  and was a dose that inhibited the growth of dermatophytes.

The LPS binding to the Toll-like receptor 4 (TLR4) triggers NF-κB nuclear translocation,  which consequently causes the expression of inducible nitric oxide synthase (Nos2) and  cycloxygenase-2 (Ptgs2) [39]. Then, we evaluated whether the EO could potentially regulate the levels of these proteins. As expected, in LPS-treated macrophages, an increase in the protein levels of both iNOS and COX-2 was observed (Figure 2A–C). The presence of S. aurea essential oil at the dose of 1.25 µL/mL could lead to a significant decrease in the protein levels of both proteins, especially iNOS. These results suggest that the essential oil might modulate the NF-κB pathway.

2.4. Wound Healing Properties of S. aurea Essential Oil

Considering the uses of S. aurea for wound treatment [18], we hypothesized that the essential oil might enhance wound healing. Indeed, our results show that the EO promoted cell migration (Figure 3A, B) without affecting cell viability (Figure 3C).

2.5. Anti-Senescence Potential of S. aurea Essential Oil

Keeping in mind that the traditional uses ascribed to S. aurea are associated with the treatment of age-related diseases [40], we assessed the effect on cell senescence. Resorting to the etoposide-induced cell senescence, we observed senescence-associated β-galactosidase activity (Figure 4A, B). Interestingly, when the essential oil was added in the recovery phase, this feature was reduced, suggesting that the essential oil appeared to prevent cell senescence.

3. Discussion

This study provides the essential oil profile of S. aurea domesticated on the island of  Sardinia. We reported that its EO was characterized by 47.4% of oxygenated monoterpenes, mainly 1,8-cineole, cis/trans-thujone, and camphor, 28.8% of hydrocarbon monoterpenes, mainly β-pinene, and 19.8% of hydrocarbon sesquiterpenes, with (E)-caryophyllene, and α-humulene, while only 2.8% of oxygenated sesquiterpenes.

When compared with the literature, a clear qualitative and quantitative difference in native S. aurea EOs composition emerges. In particular, the major components reported were α-pinene, myrcene, o-cymene, spathulenol, and α-eudesmol [32]. Moreover, these alterations are reflected in the relative percentages of the main classes of compounds. Here, we found a substantial decrease in oxygenated sesquiterpenes (2.8%), whereas they accounted for 18.9% and 41.7% in two different studies of the Western Cape region[33,41].

However, this chemical difference was not unexpected as, according to some authors, however, this chemical difference was not unexpected as, according to some authors,   it depends not only on genetic characteristics but also on a plant’s life cycle, growing season, harvest season， soil properties， and stress agents[32, 41]

In particular, about S. aurea, a consistent chemical diversity was found according to seasonality and harvest territory [32,41]. Lim Ah Tock et al., 2020, considered populations of the Western Cape area and evidenced a high degree of intraspecific variability, accounting for 47.8% [32,33,41]. Furthermore, they analyzed the HCA dendrogram of these populations,  where a clear cluster separation appeared in S. aurea, to be responsible for the substantial chemical variation [41].

In addition to the mentioned factors that impact EO chemical composition, it is known that post-harvest treatment, storage conditions, and extraction methods also contribute to the chemical variability [42,43]. With this in mind, the differences that were observed here might be attributed to any of these factors.

The reported biological activities confirm the traditional uses attributes of S. aurea,  particularly those associated with inflammatory and microbial infections.

We report that the EO inhibited the growth of dermatophytes, thus validating the use ascribed for the treatment of skin ailments [18]. The S. aurea essential oil is known to be effective against Brevibacterium [44], Staphylococcus aureus, Klebsiella pneumoniae, Bacillus cereus, and Escherichia coli [32,33]. Other studies reported antimicrobial activities for nonvolatile extracts [14,45,46]. Regarding the major compounds found in S. aurea EO, it was reported that 1,8-cineole presented weak activity against the tested dermatophytes [47]. The inhibition of plant pathogens was also reported for 1,8-cineole [48–51]. The antimicrobial potential of β-pinene was also widely reported against several yeasts and filamentous fungi,  including Trichophyton spp. [52–56]. Cis-thujone, a relevant compound in this essential oil, also exerted antimicrobial activity [57,58]. On the other hand, weak antimicrobial activity was reported for the camphor [59–61]. The sesquiterpene β-caryophyllene exerted antimicrobial activity against different fungi and bacteria [3,62–64]. These results suggest that β-pinene, cis-thujone, and β-caryophyllene might be major contributors to the activity reported for S. aurea.

Due to the traditional use of S. aurea in the treatment of inflammatory-related ailments [18], we aimed to validate these effects. Indeed, our results show that the EO  inhibited the production of NO in LPS-stimulated macrophages and concomitantly reduced the protein levels of iNOS and COX-2: two pro-inflammatory enzymes relevant to the inflammatory response. To the best of our knowledge, only one study addressed the anti-inflammatory potential of S. aurea by showing the inhibitory capacity of lipoxygenase-5 activity [33]. Regarding isolated compounds, several studies reported the anti-inflammatory activity of 1,8-cineole [65–70], including clinical trials [71]. Furthermore, it is known that this compound inhibits COX-2 activity [66] and prevents the nuclear translocation of NF-κB [65,72]. β-pinene significantly decreased NO production in both LPS-stimulated macrophages [73] and IL-1β-activated chondrocytes [74]. It also showed a chemotaxis inhibition in neutrophils [75]. Camphor’s anti-inflammatory potential is widely reported using both in vivo and in vitro models [76–78]. β-Caryophyllene also exerted anti-inflammatory effects in colitis [79], skin wound excisions [80], and sepsis [81] in animal models. Other in vitro and in vivo approaches highlight the anti-inflammatory action of this sesquiterpene [82,83]. The results suggest that the activity observed in S. aurea could be due to the presence of these compounds, which might act synergistically.

We reported that the essential oil from S. aurea promoted wound healing, which validated its uses for the treatment of open wounds. To the best of our knowledge, no studies have reported the wound-healing properties of S. aurea. Regarding the isolated compounds, 1,8-cineole promotes cell migration in wound models both in vivo and in vitro [70,84,85]. Furthermore, it was reported that essential oil from Teucrium polium subsp. capitalism, rich in β-pinene, increased wound healing [86]. The sesquiterpene β-caryophyllene also promoted wound healing in a skin wound excision model [80]. Nevertheless, other compounds found in lower amounts could contribute to the reported activity. Indeed, α-pinene promotes wound healing in the animal model of an open wound [84]. Considering that essential oils are complex mixtures, it is conceivable that the reported wound-healing properties might be attributed to the synergic effects of several compounds.

We report, for the first time, an anti-senescence effect for S. aurea essential oil. Indeed,  cells treated with the essential oil presented a reduced number of senescence-associated β-galactosidase-positive cells. Regarding isolated compounds, the number of studies reporting their effect was scarce. Indeed, camphor prevents the increased activity of senescence-associated β-galactosidase [87]. On the other hand, 1,8-cineole, the major compound of this essential oil, induced cell senescence [88]. Considering the scarcity of studies, we can only hypothesize that the reported activity might be due mainly to the camphor compound; however, the effect of minor compounds cannot be discarded.

【For more info: david.deng@wecistanche.com / WhatApp:86 13632399501】