Changes in Essential Oil Content and Composition of Salvia Limbata C.A. Mey at Different Growth Stages and Altitudes

The present study investigates the effect of growth stage (vegetative, owering and seed ripening) and altitude (1500, 2000 and 2500 m above sea level) on the content and chemical composition of S. limbata essential oil which belongs to Lamiaceae family. Results According to the oil analysis, 28 components representing 96.5% to 99.7% of the total volatile oil composition were characterized. The main compounds of S. limbata oils were α-pinene (14.7-38.7%), β-pinene (12.5-26.2%), allo-aromadendrene (9.2-21.7%), germacrene D (4.2-8.3%), bicyclogermacrene (6.5-14.5 %), and spathulenol (7.5-25.4 %). The obtained results showed that the content and constituents of S. limbata essential oil strongly depend on the growth stage and altitude. Our ndings revealed that the vegetative stage at 1500 m is the optimal harvest time to obtain the highest content of oil yield. Results of the current study helps to nd the optimum situation to gain the highest content of S. limbata essential oil but more researches are needed.


Introduction
Salvia is the largest and prominent genus of the Lamiaceae family, which includes more than 900 medicinal and ornamental species distributed in the world [1]. This genus is found in Central and South America, Western Asia and Eastern Asia [2]. Fiftyeight species of genus Salvia are found in Iran which seventeen species are endemic [3,4]. Salvia limbata C.A. Mey, a native plant of Iran, is a perennial, herbaceous and aromatic plant (30-60 cm tall) with thick, rounded and bright green leaves. The distribution of this plant within Iran is in Azerbaijan, Lorestan, Shiraz, Kermanshah, Semnan, and Damavand [5]. The genus Salvia has always been noticeable in doing research around the world for diverse biological activities and compounds in its essential oil [6,7]. Since the species of genus Salvia contain substantial amounts of essential oils, people have been applied for thousands of years in folk medicine to improve health and treat diseases [8,9]. Modern science illustrates that Salvia essential oils improve memory and could be effective in treating Alzheimer's in the future [10]. Salvia has also been used for treating coughs, colds and wounds and it has been considered as spasmolytic, antiseptic, astringent, and liver protective [11].
Moreover, the phenolic compounds of plants belonging to this genus have shown antiviral, antibacterial, antifungal, antioxidant, antitumor, antidiabetic, anxiolytic, sedative, and anti-in ammatory activities [12,13,,14,15,16,17,18]. Active compounds such as hydrocarbon monoterpenes, hydrogenated monoterpenes, oxygenated monoterpenes, di-terpenes, hydrocarbon sesquiterpenes and oxygenated sesquiterpenes have been obtained from Salvia species [19]. The contents of these compounds are in uenced by various factors such as planting conditions, harvest time, organ used and growth stage. It seems that ecological factors and genetic are the main signi cant factors that in uence the content of the plant active compounds [20].
To the best of our knowledge, few investigations have been done in the eld of autecology and phytochemistry of S. limbata. Since the global approach has been perusing the use of medicinal herbs and natural compounds in the pharmaceutical, cosmetic and food industries, there is a strong need to delve more into the issue and do further research to understand how to increase the yield of active ingredients in varied ecological conditions. This could be economically important for the food, cosmetic and pharmaceutical industries. Although phytochemistry of different species of this genus has already been studied in different ecological conditions (mostly in owering stage), for the rst time the effect of different phenological stages and altitudes on the essential oil content and composition of S. limbata was investigated in this paper.

Plant material
Aerial parts of S. limbata at several developmental stages (vegetative, owering and seed ripening) were harvested in three replicates from its wild habitat at altitudes of 1500, 2000 and 2500 m above sea level from Taleghan rangeland (semi-humid) in Alborz province, Iran (36° 5' 19" N to 36° 19' 19" N and 50° 36' 43" E to 50° 53' 20" E) (Fig. 1). Taleghan is one of S. limbata's main sites with mean relative humidity about 12%, and the average annual temperature of 11.4 o C. In this area, there are 150 freezing days and annual precipitation is about 446 mm. Harvested plant materials were dried in the shade and then ground in a grinder (2mm mesh size). A voucher herbarium specimen (MP-300) was lodged at the Herbarium of Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran.

Isolation of the essential oils
Samples (100 g) of the air-dried aerial parts of S. limbata were subjected to hydro distillation using a Clevenger-type apparatus for 4h, according to the method recommended in British Pharmacopoeia. The distilled oils were dried over anhydrous sodium sulfate and stored at 4 o C in tightly closed dark vials to be analyzed.

Chemical composition of the essential oils
For characterization of the volatile oil constituents, samples were subjected to gas chromatography-ame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS). GC-FID analysis was performed by a Shimadzu 15 A gas chromatograph (Dyson Instruments, Newcastle, U.K) equipped with a split/splitless injector (250°C), a DB-5 (60 m length × 0.25 mm internal diameter, 0.25 μm lm thickness) capillary column and ame ionization detector (250°C). The helium gas was used as the carrier gas (1 mL/min) and the oven temperature was 60-280°C at a rate of 5°C/min. The injector temperature was 250°C and 1μL of oil sample was injected in the split mode with the split ratio of 100:1.
GC-MS analysis was carried out using a Thermo-Trace GC-MS system (Thermo Electron; San José, CA, USA). This device was equipped with a DB-5 silica column (60 m length × 0.25 mm internal diameter, 0.25 μm lm thickness) containing 5%-phenyl 95%methyl polysiloxane and a mass spectrometer detector (MS). The carrier gas was helium at a ow rate of 1 ml/ min. The column temperature was kept at 60°C for 3 min and then programmed to 250°C at a rate of 5°C/min. The injector and GC/MS interface temperatures were 290°C and 300°C, respectively. The mass spectra were taken at 70 eV in the scan range of m/z 50-550.

Identi cation of compounds
The constituents of the essential oils were recognized by calculation of retention indices for all the components, using retention times of C 6 -C 24 n-alkenes series as standards under the same chromatographic conditions. Identi cation of individual compounds were performed by comparison of their retention indices and mass spectral fragmentation patterns with those reported in the literature, Wiley library (New York, NY, USA) or published mass spectra [8,21]. The relative percentage of essential oil constituents were obtained from the GC-FID peak areas in the chromatogram without the use of correction factors.

Statistical analysis
The statistical analyses were performed using Graph Pad Prism software (San Diego, CA; version 5.0). Data are presented as mean ± standard deviation (SD) in 5 randomized replicates. One way analysis of variance (ANOVA) and Tukey post-test were used to analyze obtained results. P-value < 0.05 was considered as statistically signi cant difference.

Results And Discussion
As shown in Figure 2, comparison of the essential oil yield among different samples revealed that the highest content of essential oil belongs to the harvested aerial parts of S. limbata in the vegetative stage at an altitude of 1500 m (0.86% v/w) while no signi cant difference was observed in the essential oil content among other groups. In a published study authors observed the signi cant impact of different altitudes and phenological stages on the essential oil yield [22,23]. It was revealed that plant performance is strongly in uenced by various factors such as altitude, climate, soil, developmental stages, extraction and analysis methods, genetic factors, abiotic stresses, and slope and modeling techniques can predict these factors in other areas [24,25,26,27,28]. The results of the current study were compatible with other studies that found the highest content of essential oil of Origanum majorana in the vegetative stage, so they proposed the vegetative stage as the best stage to harvest Origanum majorana [29,30,31,32,33,34,35,36].Similar results were also found byon the essential oil content of Nepeta kotschyr [37]. Moreover, according to a study conducted by the highest yield of essential oil in Teucrium polium L. was obtained in the vegetative stage [38]. These results are in agreement with our ndings. The accumulation of essential oil in vegetative stage could be due to the fact that plant protection is supplied by phenolic components which are in high amount in this stage [39].
On the contrary, it was reported bythat the highest essential oil in Satureja mutica is acquired in the owering stage [40]. In another study [41] determined that the highest value of essential oil of Mentha pieperata in owering stage, which contradicts our ndings. Also, the percentage of essential oils in vegetative stage in Thymus vulgaris was the lowest and it rose in owering stage [42]. One explanation for the increase in essential oil content in the owering stage is the maintenance of the reproductive stage [43,44] and to attract insects for pollination [35]. Nevertheless, other researches have illustrated that the lowest amount of essential oils in the vegetative stage could be due to the lower activity of some enzymes in synthesizing phenolic compounds in this stage [45]. Since photosynthetic products accumulate in the endosperm during plant growth, it leads to a decrease in the amount of essential oil [46]. It is clear that phenological stages have a great impact on the essential oil metabolism, enzymatic activity and nally essential oil content [45].
Comparing of the monoterpenes and sesquiterpenes contents of the S. limbata essential oil at different altitudes and phenological stages in Figure 4, the amount of monoterpenes has decreased from vegetative stage to seed ripening stage; however, the obtained results for sesquiterpenes were reverse. These ndings for Artemisia herba-alba essential oil were previously observed in another study [49]. Moreover, during the developing plants the amount of sesquiterpenes increased in Cannabis sativa L. which are in line with our results [50]. As we found out in our research, the highest amount of monoterpenes was related to the vegetative period at 2000 m, while the highest amount of sesquiterpenes was obtained in seed ripening stage at altitudes of 1500 and 2500 m. [51] 2002 in a research on Thymus vulgaris at different growth stages con rmed that the highest content of the monoterpene was related to the vegetative stage.
As shown in Figure 5   Data are presented as mean ±SD; RI indicates retention indices relative to C 6 -C 24 n-alkanes; Tr indicates trace (<0.1%). Data are presented as mean ±SD; RI indicates retention indices relative to C 6 -C 24 n-alkanes; Tr indicates trace

Conclusion
The data presented in this paper con rmed that the essential oil yield and constituents of S. limbata were highly in uenced by phenology and altitude. A signi cant difference in the composition of S. limbata essential oil at different growing stages and altitudes was observed. It is noteworthy that the dominant compounds of S. limbata essential oil were α-pinene, β-pinene, alloaromadendrene, germacrene-D, bicyclogermacrene, and spathulenol. Since higher yield of essential oil was obtained in vegetative stage at 1500 m, this stage could be considered as the best stage to harvest plant. Moreover, it was revealed that the highest content of monoterpenes and sesquiterpenes could be obtained at the vegetative stage and ripening stage, respectively. This was the rst research on the yield and constituents of S. limbata essential oil at different developmental stages and altitudes that could be economically bene cial for the food and pharmaceutical industries as well as other researchers to examine further investigations about this plant.

Declarations
Ethic approval: Not applicable

Consent for publication: Yes
Availability of data and materials: The form of material is in Excel le. The location was in Taleghan rangeland.