Extinction Risk Assessment and Chemical Composition of Aerial Parts Essential Oils from Two Endangered Endemic Malagasy Salvia Species

Seven essential oil samples of two endemic species of Malagasy sage, Salvia sessilifolia Baker and Salvia leucodermis Baker, were investigated via GC(RI), GC-MS and 13C NMR spectrometry. In total, 81compounds were identified accounting for 93.5% to 98.7% of the total composition. The main constituents for the both species were (E)-β-caryophyllene (29.2% to 60.1%), myrcene (1.2% to 21.7%), α-humulene (5.2% to 19.7%), (E)-nerolidol (0.8% to 15.5%) and caryophyllene oxide (1.4% to 10.8%). Ethnobotanical survey of 46 informants revealed that decoctions of leafy twigs and chewed leaves were usually used. Due to the repeated fires, over-harvesting and grazing, the populations of S. sessilifolia and S. leucodermis are drastically fragmented. These risk factors led to threats to the habitats of the target species. Salvia sessilifolia Baker and Salvia leucodermis Baker are proposed to be classified as endangered species.


Introduction
One of the largest plant families in the world, Lamiaceae contains 7173 species belonging to 236 genera [1]. Salvia (Sage) is one of these genera, and comprises 980 species distributed in tropical and temperate regions. Recent phylogenetical studies have split this genus into four clades and five additional genera. However, Malagasy species remain within the Salvia genus and represent six endemic species [2].
In the phylogenetic study at the level of the genus Salvia worldwide [2], the endemic species S. sessilifolia and S. leucodermis are closely related to species from East Africa. Indeed, subclade I-A contains two species from East Africa (S. nilotica and S. somalensis) and all Madagascar and South African species. The clade is poorly resolved with only a few small, supported terminal clades, of which one comprises S. sessilifolia and S. leucodermis.
According to Hedge et al. (1998), Salvia sessilifolia Baker and Salvia leucodermis Baker consist of sub-shrubs up to 1.50 m high, with erect, woody stems [3]. The leaves and young twigs are aromatics. The leaves are simple, opposite and without stipules. The fruit bears an accrescent and spreading-deflected calyx.
Concerning Salvia sessilifolia Baker, the leaves are sessile, rough, ascending-erect, linear-oblong to oblanceolate, cuneate at the base, crenate and slightly revolute on the edge. The upper sides of the leaf blade are green with dense, stiff, shiny hairs; the undersides ear-oblong to oblanceolate, cuneate at the base, crenate and slightly revolute on t The upper sides of the leaf blade are green with dense, stiff, shiny hairs; the un are very densely haired, white and not glandular. The inflorescences are short, c 10 flowers, are more or less close together, and are usually purple or reddish-pur ure 1).
Salvia leucodermis Baker possesses dense, close together, silvery-white, oblon tical to obovate leaves, which are velvety on both sides but very dense on the un The petioles do not exceed 7 mm. The inflorescences bring 8 to 10 clustered flower are usually white, and rarely purplish pink. (Figure 1).

Figure 1. Salvia sessilifolia (left) and Salvia leucodermis (right).
Madagascar has 3245 species of medicinal plants, 60% of which are endemic. on the use of medicinal plants have been carried out; the family of Lamiaceae is the top 30 most cited, and its frequency of citation in relation to the surveys is 52.
In recent decades, the conservation of endemic and threatened species has more attention throughout the world. Studies on the genetic diversity of endange cies have increased in recent years due to their central importance in planning bot and ex situ conservation efforts [5]. However, chemical diversity is also important uate the influence of risk factors and the study of chemical composition of essen should be a complementary approach. To our knowledge, no data are reported con these two sage endemic species.
Ecological factors refer to habitat elements that directly or indirectly aff growth, development, reproduction, behavior and distribution of plants in the e ment. For Madagascar, the global change mainly is associated with biological fac imal factor (grazing), anthropization (fire, over-harvesting, forest degradation).
Our aim is to report, for the first time, the empirical uses and the essential oil nents of two sage Malagasy endemics species, Salvia sessilifolia and Salvia leucoder to evaluate their status of conservation. On the one hand, we have studied the po status of these two species in relation to their ecology. On the other hand, we opportunity to study the chemical composition of its EO with limited impact on ulations. In the situation that an original/interesting EO composition was found, have been a supplementary argument for its preservation. Our study was carried reference to the following research permissions delivered by the ministry of envir and sustainable development:  Salvia leucodermis Baker possesses dense, close together, silvery-white, oblong-elliptical to obovate leaves, which are velvety on both sides but very dense on the underside. The petioles do not exceed 7 mm. The inflorescences bring 8 to 10 clustered flowers, which are usually white, and rarely purplish pink. (Figure 1).
Madagascar has 3245 species of medicinal plants, 60% of which are endemic. Surveys on the use of medicinal plants have been carried out; the family of Lamiaceae is among the top 30 most cited, and its frequency of citation in relation to the surveys is 52.9% [4].
In recent decades, the conservation of endemic and threatened species has received more attention throughout the world. Studies on the genetic diversity of endangered species have increased in recent years due to their central importance in planning both in situ and ex situ conservation efforts [5]. However, chemical diversity is also important to evaluate the influence of risk factors and the study of chemical composition of essentials oil should be a complementary approach. To our knowledge, no data are reported concerning these two sage endemic species.
Ecological factors refer to habitat elements that directly or indirectly affect the growth, development, reproduction, behavior and distribution of plants in the environment. For Madagascar, the global change mainly is associated with biological factors: animal factor (grazing), anthropization (fire, over-harvesting, forest degradation).
Our aim is to report, for the first time, the empirical uses and the essential oil components of two sage Malagasy endemics species, Salvia sessilifolia and Salvia leucodermis, and to evaluate their status of conservation. On the one hand, we have studied the population status of these two species in relation to their ecology. On the other hand, we took the opportunity to study the chemical composition of its EO with limited impact on the populations. In the situation that an original/interesting EO composition was found, it would have been a supplementary argument for its preservation. Our study was carried out with reference to the following research permissions delivered by the ministry of environment and sustainable development:  Ethnobotanical survey of 46 informants revealed that the uses of these two species remain the same ( Figure 2). The decoction of leafy twigs has oxytocic and antitussive properties and helps prevent certain diseases or relieve fatigue. Chewed leaves are applied to wounds, abscesses or insect bites. Its leafy branches are also sold at medicinal plant merchants around the Ankaratra massif (ANK), namely, in Antsirabe, Ambatolampy, Faratsiho (FAR), Andranomiely and Manalalondo (ITS), and generated an over-harvesting. Ethnobotanical survey of 46 informants revealed that the uses of these two remain the same (Figure 2). The decoction of leafy twigs has oxytocic and antitussiv erties and helps prevent certain diseases or relieve fatigue. Chewed leaves are ap wounds, abscesses or insect bites. Its leafy branches are also sold at medicinal pla chants around the Ankaratra massif (ANK), namely, in Antsirabe, Ambato Faratsiho (FAR), Andranomiely and Manalalondo (ITS), and generated an overing.

Ecology and Risk of Extinction
S. sessilifolia Baker is mainly distributed around the Ankaratra (ANK) and Ibi massifs, above 1300 m altitude, on rocky slopes, on wooded grassland-bushland and in Uapaca bojeri Bail. forest while S. leucodermis Baker is found in the woode land-bushland mosaic of the Ankaratra massif and those of Andringitra (ANG 1300 m altitude ( Figure 2).
Based on 25 herbarium specimens, the results of the analysis of the parame cording to the assessment of the extinction risk of these two species are summa Table 1.

Ecology and Risk of Extinction
S. sessilifolia Baker is mainly distributed around the Ankaratra (ANK) and Ibity (IBT) massifs, above 1300 m altitude, on rocky slopes, on wooded grassland-bushland mosaic and in Uapaca bojeri Bail. forest while S. leucodermis Baker is found in the wooded grasslandbushland mosaic of the Ankaratra massif and those of Andringitra (ANG), above 1300 m altitude ( Figure 2). Based on 25 herbarium specimens, the results of the analysis of the parameters according to the assessment of the extinction risk of these two species are summarized in Table 1.  25 25 Subpopulations outside and inside of the protected areas are subject to repeated fires. In the protected Manjakatompo-Ankaratra (M-ANK) area and that of Andringitra (ANG), fires are accentuated by grazing. The subpopulations outside the protected areas are threatened by the extension of agricultural lands and also grazing.
Consequently, the subpopulations of the two-target species are severely fragmented; both extent of occurrence was estimated to be less than 100 km 2 with most (≥65%) subpopulations found outside protected areas (Table 1). Moreover, continuous decline was observed for both species in the area of occupancy and occurrence, but also in the habitat.

Chemical Composition of Essential Oils
The yields calculated (Table 2) from fresh material (w/w) easily discriminated the two species: 0.11-0.23% (S. sessilifolia) vs. 0.31-0.41% (S. leucodermis). The chemical composition of the seven oil samples collected in four locations was dominated by sesquiterpene hydrocarbons. Among them, (E)-β-caryophyllene (29.2-60.1%) was the major compound (Tables 3 and 4). The chromatogram of the Sle1 sample (non-polar column) along with the main components is presented in Figure 3.

Plant Material
Samples were collected outside of the protected areas of Manjakatompo-Ankaratra and Andringitra. The data related to the sampling were reported in Figure 2 and Table 4. Vouchers were deposited in the herbarium of medicinal plants of Madagascar (CNARP). Botanical identification was carried out by the first author based at the CNARP herbarium.

Extinction Risk Assessment
The extinction risk assessment is based on the UICN Red List criteria for plant species, version 3.1 [21]. Geographic range (criteria B) was used to estimate the extinction risk. Geographical localities of previous collections of the two species were consulted in the (i) herbarium of medicinal plants of Madagascar (CNARP), (ii) online herbarium of the MNHN, (iii) herbaria of the Botanical and Zoological Park of Tsimbazaza and (iv) Tropicos database site.
The number of the sub population (geographically or otherwise distinct groups in the total number of individuals of the taxon between which demographic or genetic exchange are very little), extent of occurrence (the area limited within the shortest continuous imaginary boundary which can be drawn to encompass all the known, inferred or projected sites of present occurrence of a taxon, excluding cases of vagrancy) and area of occupancy (the area occupied by a taxon within its extent of occurrence, excluding cases of vagrancy) were determined by using Arc view software. Threat or Declin population (DP) were calculated according to the formula: Botanical surveys were carried out in the massif of Ankaratra and its surroundings and in that of Andringitra to obtain the data on distribution, in order to know the states (E)-β-caryophyllene, was reported as a main component for several Salvia species from Colombia, Iran, Turkey and Spain [14]. However, the chemical compositions of S. sessilifolia and S. leucodermis were drastically different from those of East African species [15][16][17][18][19][20] (Table S1). Among the sixteen chemical compositions reported in the literature for 15 species; the percentage of (E)-β-caryophyllene ranged from 0 to 13.1%, and the major components were the mainly oxygenated compound: 1,8-cineole (40.5%; S. chamelaegnea) [19], linalool (44.4%; S. schimperi) [16], geraniol (19.6%; S. dolomitica in South Africa), linalyl acetate (19.6%; S. dolomitica in South Africa) [20], bornyl acetate (16.2%; S. somaliensis) [17], caryophyllene oxide (22.6%; S. radula) [19], spathulenol (29.1%; S. africana-caerulea) [19], viridiflorol (24.5%; S. albicaulis) [20], and α-bisabolol (65.5%; S. runcinata) [20].

Plant Material
Samples were collected outside of the protected areas of Manjakatompo-Ankaratra and Andringitra. The data related to the sampling were reported in Figure 2 and Table 4. Vouchers were deposited in the herbarium of medicinal plants of Madagascar (CNARP). Botanical identification was carried out by the first author based at the CNARP herbarium.

Extinction Risk Assessment
The extinction risk assessment is based on the UICN Red List criteria for plant species, version 3.1 [21]. Geographic range (criteria B) was used to estimate the extinction risk. The number of the sub population (geographically or otherwise distinct groups in the total number of individuals of the taxon between which demographic or genetic exchange are very little), extent of occurrence (the area limited within the shortest continuous imaginary boundary which can be drawn to encompass all the known, inferred or projected sites of present occurrence of a taxon, excluding cases of vagrancy) and area of occupancy (the area occupied by a taxon within its extent of occurrence, excluding cases of vagrancy) were determined by using Arc view software. Threat or Declin population (DP) were calculated according to the formula: Botanical surveys were carried out in the massif of Ankaratra and its surroundings and in that of Andringitra to obtain the data on distribution, in order to know the states of the habitats, and to determine the threats to the species and its uses. Voucher specimens were made during the fieldwork and deposited in the herbarium of medicinal plants of Madagascar.
During the botanical surveys, the information on traditional uses were collected from 46 persons: 26 around the Ankaratra massif and 20 on the northeastern slope of the Andringitra massif.

Essential Oil Isolation
For each species, individual samples (aerial parts) were collected in a limited area, early in the morning and in dry weather. Aerial parts (200 g) were submitted to hydrodistillation for 2 h 30 with a Clevenger-type apparatus in a 1 L flask. The yields were calculated from fresh material (w/w) ( Table 2). The essential oil samples obtained were conserved at 4 • C.

Gas Chromatography and Gas Chromatography-Mass Spectrometry in Electron Impact Mode
GC analyses were performed on a Clarus 500 PerkinElmer Chromatograph (PerkinElmer, Courtaboeuf, France), equipped with a flame ionization detector (FID) and two fused-silica capillary columns (length, 50 m; diameter, 0.22 mm; film thickness, 0.25 µm), BP-1 (polydimethylsiloxane) and BP-20 (polyethylene glycol). The oven temperature was programmed from 60 • C to 220 • C at 2 • C/min and then held isothermal at 220 • C for 20 min; injector temperature: 250 • C; detector temperature: 250 • C; carrier gas: hydrogen (0.8 mL/min); split: 1/60; injected volume: 0.5 µL. The relative proportions of the oil constituents were expressed as percentages obtained by peak-area normalization, without using correcting factors. Retention indices (RI) were calculated relative to the retention times of a series of n-alkanes (C8-C29) with linear interpolation («Target Compounds» software from PerkinElmer).

Nuclear Magnetic Resonance
All nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AVANCE 400 Fourier Transform spectrometer (Bruker, Wissembourg, France) operating at 100.623 MHz for 13 C, equipped with a 5 mm probe. The solvent used was CDCl 3 , with all shifts referred to internal tetramethyl silane (TMS). 13 C NMR spectra of the oil samples were recorded with the following parameters: pulse width, 4 µs (flip angle 45 • ); relaxation delay D1, 0.1 s; acquisition time, 2.7 s for a 128 K data table with a spectral width of 25,000 Hz (250 ppm); CPD mode decoupling; digital resolution, 0.183 Hz/pt. The number of accumulated scans was 3000 for each sample (30 mg in 0.5 mL of CDCl 3 ).

Identification of Individual Components
Identification of the individual components was carried out as follows: (i) by comparison of their GC retention indices on non-polar and polar columns, with those of reference compounds [6,7,10]; (ii) on computer matching against commercial mass spectral libraries [7,22,23]; (iii) on comparison of the signals in the 13 C NMR spectra of the samples with those of reference spectra compiled in the laboratory spectral library, with the help of laboratory-made software [24][25][26]. This method allowed the identification of individual components of the essential oil at contents as low as 0.4%.

Conclusions
The essential oils of aerial parts of Salvia sessilifolia Baker and Salvia leucodermis Baker were characterized by the presence, of (E)-β-caryophyllene, which, as a major component, possesses several important pharmacological activities, ranging from pain treatment to neurological and metabolic disorders.
According to the results of the parameters assessed and the observed threats on the habitats and our botanical prospections, the target species, Salvia sessilifolia Baker and Salvia leucodermis Baker are proposed to be classified as endangered species or EN B2ab(ii, iii, iv).

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants12101967/s1, Table S1. Main components of essential oils of Salvia from East Africa.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.