Essential Oil Composition and Traditional Uses of Salvia dentata, a Poorly Known Medicinal Plant from Namaqualand, South Africa

South Africa has a rich history of medicinal plant species and their documented uses as traditional medicines, and is also home to three well-known, blue-flowered sage species of ethnobotanical importance. The Namaqualand bloublomsalie (Salvia dentata) has so far remained unstudied and apparently overlooked. Our study is the first to report on the essential oil chemistry of this medicinally relevant species and provide a comparison with the other two (well-studied) closely related Cape bloublomsalies (Salvia africana and S. chamelaeagnea). The data, generated from three geographically isolated populations comprised of 13 individual plants of S. dentata, revealed diagnostically high levels of camphor (14.37%), α-pinene (11.43%), camphene (10.18%), 1,8-cineole (eucalyptol) (9.42%) and bornyl acetate (8.56%) which provide a distinct chemical profile from the other two species.


Introduction
The genus Salvia L. is the largest genus within the Lamiaceae (ca. 1010 spp. [1]) with various species used worldwide in traditional and folk medicines, treating ailments such as angiogenesis, bacterial and viral infections, inflammation and oxidative stress [2]. Although the genus was recognized by both Egyptian and the Greek civilizations, the name Salvia is derived from the Latin word "salvēre", meaning "to feel healthy, to heal" given by the Roman Empire [3,4]. Many members of this genus are well known, such as S. officinalis L. (common sage), S. rosmarinus Spenn. (rosemary; hitherto better known as Rosmarinus officinalis L. [5]), S. officinalis subsp. lavandulifolia (Vahl) Gams (Spanish sage) and S. fruticosa Mill. (Greek sage) [6], largely for their therapeutic effects owed to their bioactive constituents [7]. These constituents include compounds such as 1,8cineole (eucalyptol), α-thujone, camphor and camphene found in their essential oil (EO), as well as phenolic compounds such as caffeic-, rosmarinic-and salvianolic acid. In terms of flavonoids, 6-hydroxyflavones are characteristic of Salvia species and can be used as a chemosystematic/chemophenetic marker [8]. Being the largest genus within the Lamiaceae, the species have a wide geographical distribution, with the greatest concentration in Mexico and the Mediterranean region. In Asia, more than 40 species of Salvia are indigenous to China, with S. miltiorrhiza Bunge (red sage) being a popular herb in traditional Chinese medicine locally referred to as 'Danshen' or 'Tanshen' which is used to treat cardiovascular and renal ailments [7]. Salvia hispanica L., native to Mexico and Ecuador, is used both medicinally and as a source of protein where the seeds of this species are used as the notable superfood popularly known as 'chia' [9]. 2

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Southern Africa is home to 28 Salvia species, 14 of which are endemic to the region [10]. The Cape region of South Africa houses, amongst others, three closely related species of blue-flowered sages that are used in traditional medicines and are locally referred to as bloublomsalies ("blue flower sages"), and are Salvia africana L., S. chamelaeagnea Berg. and S. dentata Aiton ( Figure 1A-C). The trichomes (hairs and glands) on the calyces are of diagnostic value used to distinguish between the three species [11]. Salvia africana and S. chamelaeagnea have been well studied, both in terms of their ethnobotanical records, pharmacological effects and chemistry [12,13]. The third species however, which is centered in Namaqualand ( Figure 2)-is less well known and requires further investigation. Some traditional uses of S. africana and S. chamelaeagnea include the treatment of coughs, colds, influenza, chest troubles, headaches and fever [14,15]. Salvia dentata is used for the treatment of coughs, colds, influenza and stomach complaints [16,17]. Furthermore, an overlap exists between the traditional uses of these three medicinal Salvia species by various cultural groups of the Western and Northern Cape provinces, especially in the treatment of respiratory and gastrointestinal ailments. No publications could be found regarding the chemistry and essential oil composition of S. dentata. The aim of this paper is to present a review of all recorded ethnobotanical information about these three medicinally important species and to report for the first time the volatile components of S. dentata and compare the essential oil chemistry to that of the other two well-studied species.
Molecules 2022, 25, x FOR PEER REVIEW 2 of 30 Ecuador, is used both medicinally and as a source of protein where the seeds of this species are used as the notable superfood popularly known as 'chia' [9]. Southern Africa is home to 28 Salvia species, 14 of which are endemic to the region [10]. The Cape region of South Africa houses, amongst others, three closely related species of blue-flowered sages that are used in traditional medicines and are locally referred to as bloublomsalies ("blue flower sages"), and are Salvia africana L., S. chamelaeagnea Berg. and S. dentata Aiton ( Figure 1A-C). The trichomes (hairs and glands) on the calyces are of diagnostic value used to distinguish between the three species [11]. Salvia africana and S. chamelaeagnea have been well studied, both in terms of their ethnobotanical records, pharmacological effects and chemistry [12,13]. The third species however, which is centered in Namaqualand ( Figure 2)-is less well known and requires further investigation. Some traditional uses of S. africana and S. chamelaeagnea include the treatment of coughs, colds, influenza, chest troubles, headaches and fever [14,15]. Salvia dentata is used for the treatment of coughs, colds, influenza and stomach complaints [16,17]. Furthermore, an overlap exists between the traditional uses of these three medicinal Salvia species by various cultural groups of the Western and Northern Cape provinces, especially in the treatment of respiratory and gastrointestinal ailments. No publications could be found regarding the chemistry and essential oil composition of S. dentata. The aim of this paper is to present a review of all recorded ethnobotanical information about these three medicinally important species and to report for the first time the volatile components of S. dentata and compare the essential oil chemistry to that of the other two well-studied species.

Morphological characteristics
Although the three species of Salvia appear to be morphologically similar, subtle differences exist by which the species can be distinguished from each other. The most useful and obvious diagnostic character is the pubescence of the calyx. The following characters and character states have been extracted from Codd [11] and can be used in identifying each species: 2.1.1. Salvia dentata A twiggy erect shrub ca. 2 m tall with greyish-tomentulose, gland-dotted stems ( Figure 3A). Inflorescence of 2-9 spaced or crowded verticils, 2-6-flowered ( Figure 3B-C).

Morphological Characteristics
Although the three species of Salvia appear to be morphologically similar, subtle differences exist by which the species can be distinguished from each other. The most useful and obvious diagnostic character is the pubescence of the calyx. The following characters and character states have been extracted from Codd [11] and can be used in identifying each species: 2.1.1. Salvia dentata A twiggy erect shrub ca. 2 m tall with greyish-tomentulose, gland-dotted stems ( Figure 3A). Inflorescence of 2-9 spaced or crowded verticils, 2-6-flowered ( Figure 3B,C). The calyx is somewhat funnel shaped, hispid and generally copiously red gland-dotted and occasionally hispid-villous ( Figure 3D). The corolla is light blue or whitish to violet-blue to purple ( Figure 3B-D). The calyx is somewhat funnel shaped, hispid and generally copiously red gland-dotted and occasionally hispid-villous ( Figure 3D). The corolla is light blue or whitish to violetblue to purple ( Figures 3B-D).

Salvia Africana
A shrub ca. 2 m tall which often branches at the base ( Figure 4A) with several erect, usually sparingly branched stems of which are greyish-tomentulose to hispidulous and gland-dotted with occasional glandular hairs. Inflorescences are often dense or spaced below with 5-12 verticils which are 2-6-flowered ( Figure 4B). The calyx is somewhat funnel-shaped, glandular-villous and purple tinged ( Figure 4B). The corolla is light blue to bluish purple or pinkish with the lower lip usually with a paler blue margin and white to yellowish in the center ( Figure 4B).

Salvia Chamelaeagnea
A much-branched shrub 0.6-2 m tall ( Figure 5A) with stems that are scabrid to pilose and gland-dotted. Inflorescences are large panicles, 100-300 mm long, with verticils twoflowered ( Figure 5B). The calyx is reddish purple, glandular-hispid and gland-dotted. The corolla is blue or purplish blue often with white on the lower lip ( Figure 5C-D).

Salvia africana
A shrub ca. 2 m tall which often branches at the base ( Figure 4A) with several erect, usually sparingly branched stems of which are greyish-tomentulose to hispidulous and gland-dotted with occasional glandular hairs. Inflorescences are often dense or spaced below with 5-12 verticils which are 2-6-flowered ( Figure 4B). The calyx is somewhat funnel-shaped, glandular-villous and purple tinged ( Figure 4B). The corolla is light blue to bluish purple or pinkish with the lower lip usually with a paler blue margin and white to yellowish in the center ( Figure 4B).

Salvia chamelaeagnea
A much-branched shrub 0.6-2 m tall ( Figure 5A) with stems that are scabrid to pilose and gland-dotted. Inflorescences are large panicles, 100-300 mm long, with verticils twoflowered ( Figure 5B). The calyx is reddish purple, glandular-hispid and gland-dotted. The corolla is blue or purplish blue often with white on the lower lip ( Figure 5C,D).

Ethnobotanical Data
For the three Salvia species, the overall use categories with the highest number of userecords are respiratory system (32 records), gastrointestinal (16 records), analgesic (9 records), reproductive system (8 records) and topical applications (7 records; Figure 6). The ethnobotanical data show that Salvia dentata is mainly used for the treatment of respiratory ailments (colds, coughs and influenza), topical application, gastrointestinal (stomach complaints and diarrhoea) and unspecified medicinal uses (Table 1). Interestingly all three of

Ethnobotanical Data
For the three Salvia species, the overall use categories with the highest number of use-records are respiratory system (32 records), gastrointestinal (16 records), analgesic (9 records), reproductive system (8 records) and topical applications (7 records; Figure 6). The ethnobotanical data show that Salvia dentata is mainly used for the treatment of respiratory ailments (colds, coughs and influenza), topical application, gastrointestinal (stomach complaints and diarrhoea) and unspecified medicinal uses (Table 1). Interestingly all three of the species are documented to be used as treatment for similar ailments (colds, coughs, influenza, stomach complaints and woman's ailments) by different cultural groups across the Western and Northern Cape provinces.
In South America Salvia oppositiflora Ruiz & Pav., S. tubiflora Sm., S. dombeyi Epling and S. revoluta Ruiz & Pav. are used by indigenous people primarily for the treatment of respiratory ailments. Secondary uses include analgesic, stomach concerns and topical uses [18]. Similarly, in the East Mediterranean, S. fruticosa Mill. [=S. libanotica Boiss. & Gaill.] is a popular medicinal plant that is widely used in the region. In Lebanon, Syria and Jordan a tea is made from the leaves which are commonly sold at the market and drunk for abdominal pain, headaches, stomach complaints and several other disorders. In Palestine it used for the treatment of stomach and cardiovascular issues. Furthermore, in Turkey, the plant is used to treat colds, coughs, influenza, and for gall bladder and kidney stones [19].
There seems to be a cultural overlap regarding the ethnobotanical uses across the globe for Salvia species in the treatment for certain conditions (i.e., respiratory, gastrointestinal, and topical). leaves: leaf decoctions used for coughs, colds, women's ailments, diarrhoea [31] leaves: colds, influenza, stomach complaints, unspecified medicinal use, measles; other: firewood [17] unspecified medicinal use [23]

Chemical Data
For comparative reasons, EOs were distilled from fresh material of Salvia africana, S. chamelaeagnea and S. dentata collected from several localities within the Northern and Western Cape provinces of South Africa (Figure 2; data points A-I). The mean percentage yield of EOs obtained for each group was 1.23% for S. dentata, 0.56% for S. africana and 0.54% for S. chamelaeagnea. The mean percentage yield obtained for S. africana was higher than results reported by other studies (0.17% [12], 0.13% [32] and 0.14% [33]), whereas the mean percent yield for S. chamelaeagnea were similar to those reported by Kamatou et al. [32] (0.39%) and Lim Ah Tock et al. [34] (0.43%). Higher EO yields are likely due to factors such as vegetative state, temperature, climate, geology and time of harvest [35,36]. Furthermore, the higher yield of S. dentata EO could be as a direct result of the species having a higher density of glandular trichomes on the leaf surface, which was observed for S. africana and S. chamelaeagnea by Kamatou et al. [32]. The essential oil obtained from S. dentata, S. africana and S. chamelaeagnea presented a pale-yellow color and all of the samples were aromatic. Further, S. dentata presented a highly aromatic odor distinct from the other two species.
Essential oils were analyzed by gas chromatography (GC) and gas chromatographymass spectrometry (GC-MS). A total of 89 compounds were identified across three species (Tables 2-4

Chemical Data
For comparative reasons, EOs were distilled from fresh material of Salvia africana, S. chamelaeagnea and S. dentata collected from several localities within the Northern and Western Cape provinces of South Africa ( Figure 2; data points A-I). The mean percentage yield of EOs obtained for each group was 1.23% for S. dentata, 0.56% for S. africana and 0.54% for S. chamelaeagnea. The mean percentage yield obtained for S. africana was higher than results reported by other studies (0.17% [12], 0.13% [32] and 0.14% [33]), whereas the mean percent yield for S. chamelaeagnea were similar to those reported by Kamatou et al. [32] (0.39%) and Lim Ah Tock et al. [34] (0.43%). Higher EO yields are likely due to factors such as vegetative state, temperature, climate, geology and time of harvest [35,36]. Furthermore, the higher yield of S. dentata EO could be as a direct result of the species having a higher density of glandular trichomes on the leaf surface, which was observed for S. africana and S. chamelaeagnea by Kamatou et al. [32]. The essential oil obtained from S. dentata, S. africana and S. chamelaeagnea presented a pale-yellow color and all of the samples were aromatic. Further, S. dentata presented a highly aromatic odor distinct from the other two species.
The variability and potential diagnostic value of the essential oil data was investigated, and a principal component analysis (PCA) indicates three groupings for the three species from the first (14.70%) and second (10.40%) principal components (Figure 7). The S. dentata group shows almost no overlap with the S. africana and S. chamelaeagnea groups.
Other Salvia species reported with high levels of camphor and 1,8-cineole include S. aytachii Vural & Adigüzel [40] from Turkey and S. fruticosa from Lybia [41]. Alone, each compound has been reported to exhibit potent biological activity. For example, camphor, a natural product derived from the wood of Cinnamomum camphora L., though also present in other aromatic plant species, such as Ocimum kilimandscharicum Gürke (Lamiaceae), is a major source of the compound in Asia [42]. Camphor has been reported to be a counterirritant, rubefacient, mild analgesic, components of liniments for the relief of fibrositis, neuralgia and is a mild expectorant. Camphor oil has been used intramuscular or subcutaneously as a circulatory/respiratory stimulant though these methods are considered hazardous [42,43]. Eucalyptol (1,8-cineole) is a mucolytic monoterpene that has been shown to exhibit anti-inflammatory, bronchodilatory and antimicrobial activity and has been used effectively in the treatment of asthma and associated respiratory ailments [44].
Other Salvia species reported with high levels of camphor and 1,8-cineole include S. aytachii Vural & Adigüzel [40] from Turkey and S. fruticosa from Lybia [41]. Alone, each compound has been reported to exhibit potent biological activity. For example, camphor, a natural product derived from the wood of Cinnamomum camphora L., though also present in other aromatic plant species, such as Ocimum kilimandscharicum Gürke (Lamiaceae), is a major source of the compound in Asia [42]. Camphor has been reported to be a counterirritant, rubefacient, mild analgesic, components of liniments for the relief of fibrositis, neuralgia and is a mild expectorant. Camphor oil has been used intramuscular or subcutaneously as a circulatory/respiratory stimulant though these methods are considered hazardous [42,43]. Eucalyptol (1,8-cineole) is a mucolytic monoterpene that has been shown to exhibit anti-inflammatory, bronchodilatory and antimicrobial activity and has been used effectively in the treatment of asthma and associated respiratory ailments [44].
The higher levels of both camphor and 1,8-cineole in S. dentata may suggest to its use as a traditional remedy in the treatment of microbial infections (i.e., respiratory, gastrointestinal and topical) due to the synergistic effect of these two compounds as reported by Viljoen et al. [45].

Ethnobotanical Data
Literature searches were conducted by searching several scientific electronic databases, including GoogleScholar (www.scholar.google.com), EBSCOhost (www.ebsco.com), PubMed (www.pubmed.ncbi.nlm.nih.gov), ScienceDirect (www.sciencedirect.com), SciFinder (www.scifinder.cas.org), Springer (www.springer.com) and Wiley Online Library (www.onlinelibrary.wiley.com). Key words were used to search for literature, and this was conducted in the following manner: ("Species name" AND "synonyms" AND "med*") and ("Species name" AND "synonyms" AND "traditional use"). A collection of scientific papers, books, dissertations and theses, and unpublished sources were also compiled.

Plant Material and Isolation of Essential Oil
Fresh aerial parts of Salvia africana (n = 6), S. chamelaeagnea (n = 6), and S. dentata (n = 13), were collected from natural populations in the Cape region of South Africa ( Table 5). The three species were identified by B.-E. Van Wyk and voucher specimens are housed at the University of Johannesburg Herbarium (JRAU), University of Johannesburg, South Africa. The plant material was airdried at room temperature (indoors, at ca. 18 to 22 • C) for several days until no moisture was present. The essential oils were isolated by subjecting the dried leaf material to hydrodistillation using a Clevenger apparatus for three hours. The resulting EO was stored in amber vials at +4 • C until tested and all results were expressed based on dry matter weight.

Chemical Analyses
Gas chromatography and gas chromatography coupled to mass spectrometry-A modified method based on Boukhatem, Kameli and Saidi [46] was used where EO samples were prepared by diluting 20 µL EO in 1000 µL acetone. One microliter of the diluted sample was injected onto a Thermo Scientific™ TRACE™ 1300 gas chromatograph (Rodano, Milan-Italy) equipped with a ZB-5Ms capillary column (30 m × 0.25 mm, 0.25 µm), coupled with a Thermo Scientific™ TSQ™ 8000 Triple Quadrupole mass spectrometer (Austin, TX, USA). The GC-MS system was coupled to a Triplus RSH Analytics PAL autosampler (Switzerland). Chromatographic separation of the essential oil was performed on a non-polar ZB-5Ms (30 m, 0.25 mm ID, 0.25 µm film thickness) Zebron capillary column. Helium was used as the carrier gas at a flow rate of 1 mL/min. The injector temperature was maintained at 250 • C. A total of 1µl of the sample was injected in 50:1 split mode. The oven temperature was programmed as follows: initial temperature of 40 • C held for 5 min; and finally ramped up to 250 • C at a rate of 8 • C/min and held for 5 min with a total runtime of 36 min. The MSD was operated in a full scan mode and the source and quad temperatures were maintained at 230 • C and 150 • C, respectively. The transfer line temperature was maintained at 250 • C. The mass spectrometer was operated under electron impact mode at ionization energy of 70 eV, scanning from 35 to 500 m/z. The Salvia EOs constituents were tentatively identified by comparing their Retention Indices (RI) with those from literature such as Adams [47] and the similarity of their mass spectra with those databased in the NIST-MS library. RI were calculated under the same operating conditions in relation to a homologous series of n-alkanes (C8-C24).

Conclusions
Although the three species of blue-flowered sages are closely related, Salvia dentata presents a distinct chemical profile from the other two species (S. africana and S. chamelaeagnea) due to the higher quantities of camphene, camphor, bornyl acetate and 1,8-cineole (eucalyptol) present as highlighted by the data. For the first time, the essential oil chemistry has been reported for this medicinally relevant species and presents opportunities for further investigation within the fields of biological activity and phenolic chemistry.

Data Availability Statement:
The geographical data presented in this study are openly available in GBIF (Global Biodiversity Information Facility) at https://doi.org/10.15468/dl.sa4wq9.