Advances in Crop Science and Technology
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Chemical composition; Chemo types; Essential oil; Salvia rosmarinus; Variability

Introduction

Plant secondary metabolites, such as essential oils are recognized for their various biological effects like antimicrobial, antifungal, antibacterial, insecticidal and antioxidant activities [1] and applied in the fields of cosmetics, sanitary, pharmacology and food preservation [2]. Of essential oil-bearing plants, Salvia rosmarinus Schleid, commonly known as rosemary is ancient plant that has been widely assessed for the quality of its essential oils and traded all over the world [2, 3]. The essential oil of rosemary owns significant antimicrobial, anti-cancerous, anti-lipid peroxidation, and several other medicinal activities [4]. It also possesses high antioxidant activity and widely used as natural preservatives in the food and cosmetic industry [5, 6].

Notably, rosemary essential oil is characterized by the presence of various chemical compounds which are primarily responsible for its different biological activities and anti-oxidant properties [7]. Studies carried out elsewhere showed that chemical compositions such as α-pinene, bornyl acetate, camphor and 1, 8-cineole are among the main compounds responsible for the anti-microbial activities of rosemary essential oil [8].The biological activities of various rosemary oil chemical compositions have been also described in different literatures [9].

Due to consumers’ concern about the negative side effects of synthetic products and the growing demand for natural agents [10], rosemary extracts have received significant attention because of their strong natural antioxidant and antimicrobial properties. Thus, characterizing the chemical composition of rosemary essential oils and understanding the extent of chemical variability is crucial to exploit the available germplasm for the desired compositions.

The chemical composition of rosemary essential oil has been widely studied; andfigure 1 its high variability in relation to genetic factors, geographic origin, and isolation methods were reported. High variability in rosemary essential oil was evident by the presence of at least 13 different rosemary oil chemotypes elsewhere, based on their relative percentages of α-pinene, 1,8-cineole, camphor, borneol, verbenone, and bornyl acetate (Satyalet al., 2017). Different findings indicated that the chemical variability of rosemary essential oil is mainly dependent on the genetic factors rather than on environmental conditions and geographic locations, suggesting the possibilities of genetic manipulations to improve the crop for desired compounds.

An effective improvement program, in turn, largely depends on the prior characterization of the germplasm for the extent of genetic variability in the required characters. Although several studies were carried-out to investigate the chemical composition of rosemary essential oil in different countries, there is no effort made to examine the chemical composition of rosemary essential oil in Ethiopia except a study conducted byBekriet al. (2018) on chemotypic characterization of only three Ethiopia rosemary varieties.The current study was, therefore, designed to assess the variability exists in essential oil compositionof rosemary germplasms grown in Ethiopia.

Materials and Methods

Experimental site and plant materials

A total of 45 rosemary accessions were used for the study. Detail information of the accessions and experimental site is given in chapter 2 (sections 2.2.1 and 2.2.2). The accessions were multiplied via stem cutting to get sufficient planting materials for the experiment. After multiplication of the accessions, soft stem cuttings were taken and seedlings were raised in the nursery before being transplanted to the experimental field. After being raised in the nursery, healthy, equalsize, and well performed seedlings were transplanted in the main experimental field with inter and intra row spacing of 60 cm. Prior to chemical composition analyses, all accessions were cultivated for more than 2 years (from seedling multiplication to field experimentation) under homogeneous field management conditions to minimize the influence of environmental factor on chemical composition of rosemary essential oil as suggested by Li et al. (2016).

Sample preparation and essential oil extraction

For essential oil extraction, fresh leaves of composite samples (300 g) was taken from each accession and subjected to hydro-distillation for 4 hrs using a Clevenger-type apparatus. Then essential oil was collected after drying with an anhydrous Na2SO4, and stored in a refrigerator until GC-MS analysis.

Gas chromatography-mass spectrometry (GC-MS) analysis

GC-MS analysis was carried out in the Natural Products Laboratory at Wondo Genet Agricultural Research Center. The identification of the essential oil composition was performed using a GC-MS (Agilent model 7820A) equipped with auto sampler (Agilent model G4513A), MS detector (5975) and HP-5ms capillary column (0.25 mm i.d. × 30 m × 0.25 μm film thickness). A 0.2% (w/v) of the essential oil solution was prepared by diluting in n-hexane and the instrument was conditioned with a split less injector mode. Oven temperature of GC was programmed as: initiation at 60°C for 1 min, increase to 80oC at 5oC/min intervals, which were kept for 3 min, and increased to 180oC at 4oC/ min interval and held for 3 min and finally increased to 300oC at 25oC/min interval with a holding time of 6min. The injection part temperature was set at 250°C. Helium was used as carrier gas and controlled in constant flow mode at a linear velocity of 36.6 cm/sec. The mass spectrometer interface temperature was set at 260°C and operated on scan mode in 40-500 m/z range, with ion source and transfer line temperatures of 230°C and 260°C, respectively. Injection of the sample was operated on a split ratio of 1:5 with an injection volume of 1μL. The GC-MS total run time was 46 min with solvent delay time of 3.4 min. The identification of volatile compounds was primarily based by comparing their mass spectra, performed with MSDChem software (Agilent), with NIST/WILEY libraries, and retention times.

Statistical analysis

Compounds that were not detected in the study were given a value of zero. Twelve compounds at levels more than 1.5% were used for analysis and discussion. The compounds were: α-pinene, 1,8-cineole, camphor, verbenone, β-caryophyllene, endo-borneol, camphene, bornyl acetate, limonene, α-terpineol, β-pinene and linalool. Descriptive analysis using MINITAB version 17 software was conducted and percentage of compounds across accessions were presented as mean ± SE. Correlations analysis among the major compounds was performed using the same software

Results and Discussion

Chemical composition of the essential oils

GC-MS analysis of the essential oil extracted from the 45 rosemary accessions allowed identification of forty-two compounds, accounting for 95.85-98.89% of the total oil composition. The main constituents identified in most of the accessions were α-pinene (5.08 - 40.62%), 1,8-cineole (8.13 - 38.48%), camphor (2.15 - 23%), verbenone (1.83 - 20.25%), β-caryophyllene (2.12 - 9.39%), endo-borneol (1.79 - 12.56%), camphene (1.69 - 7.86%,), bornyl acetate (1.55 - 9.65%), limonene (1.65 - 6.07%), α-terpineol (1.66 - 6.37%), β-pinene (1.55 - 6.45%) and linalool (1.58 - 3.91%). The level of identified compounds revealed the presence of high chemical variability among rosemary accessions grown in Ethiopia.

It was interesting to note that α-pinene, 1, 8-cineole, camphor, and verbenone dominated the essential oil composition, and were found to present in all accessions, while the rest varied among the accessions. Previous studies explained that the presence of α-pinene, 1, 8-cineole, camphor and verbenone as the major characteristic components of this species, which is in consistent to our data. In fact, several biological activities of rosemary oils were also attributed to the presence of these major compounds (Table 1).

Table 1: Retention times range and mean of the 12 main essential oil constituents across 45 rosemary accessions.

Among the major compounds, α-pinene was the most ubiquitous constituent varied from 5.08 (Ros41) to 40.62% (Ros38) across accession with an average value of 24.57% .Congruent to this result several studies reported an abundant distribution of α-pinene in rosemary genotypes elsewhere. 1,8-cineole was the second most abundant constituent found in the range of 8.13% (Ros23) to 38.48% (Ros33) between accessions with an overall mean value of 18.88% an outcome similar to that described by different authors for rosemary from various areas.

The relative quantities of camphor and verbenone ranged between 2.15% (for Ros34) and 23% (for Ros29); and 1.83% (for Ros21) and 20.25% (for Ros24), respectively. In agreement with this finding, wide variation in camphor (7.27–13.02%) and verbenone (0.15–6.61%) contents of different rosemary varieties essential oil were reported. It is also important to mention the existence of some accessions with an essential oil rich in endo-borneol as much as: 10.58% (Ros05), 11.97% (Ros17), 12.56% (Ros18), and 10.11% (Ros34). The result obtained here is in accordance with an earlier study by Bekriet al. (2018), who defined an abundant presence of α-pinene, 1,8-cineole, camphor, verbenone, and endo-borneol in the essential oil of Ethiopian rosemary verities.

Rosemary essential oils are known to have different biological activities which depend on their chemical composition, and the different compositions are likely to present different biological activities. Concerning this, different researchers reported antimicrobial and antioxidant activity of rosemary essential oil rich in 1,8-cineole, camphor, and borneol. Similarly, strong antimicrobial activity of rosemary oil rich in α-pinene, bornyl acetate, camphor, and 1, 8-cineolewas described at different times. Several biological activities of 1, 8-cineol, α-pinene, and camphor from rosemary essential oil was also stated by different authors elsewhere. Thus, the major compounds detected in the essential oil of the studied accessions indicated the richness of the oil for various biological activities.

Besides, rosemary essential oil has wider application in cosmetics and food industries. Hence the higher essential oil composition variability observed in the tested accessions lies with the great potential of Ethiopian rosemary accessions for broader application either by direct selection or through improvement activities (Figure 1).

Figure 1: GC-MS chromatogram of Ethiopian rosemary essential oil.

Correlation among the major constituents

In order to investigate the relationship among chemical variables, correlation analysis was performed (Table 6.2). Examining the association among the major compounds, α-pinene correlated negatively with camphor (r = -0.462***), endo-borneol (r = -0.632***), α-terpineol (r=0.328*), verbenone (r=-0.405**), bornyl acetate (-0.51***) and β-caryophyllene (-0.41**), and positively with camphene (r=0.434**). Similarly, the association of 1, 8-cineole with linalool (r=-0.321*), endo-borneol (r=-0.434**), α-terpineo (r=- 0.528***), verbenone (r=-0.357*), bornyl acetate (r=-0.52***) and β-caryophyllene (r= -0.54***) was negative. Verbenone correlated negatively with camphene (r=-0.308*) and β–pinene (r=-0.467***), and positively with α-terpineol (r=0.310*) and bornyl acetate (r=0.33*). The association of camphor with the entire major constituent was nonsignificant except its negative association with α-pinene (r= -0.46***) and linalool (r=-303*). 1, 8-cineole and α-pinene doesn’t correlated to each other (r = 0.143ns), but each of them strongly correlated to the other constituents, creating variable chemical groups.

Understanding the relationship among the main chemical constituents would give insight to the breeders on how to manipulate and improve the crop for the desired chemical constituents. In this study, it is observed that α-pinene and camphene correlate positively to each other, showing an increase in one of them might result in an increase of the other. On the other hand, a significant negative correlation of α-pinene, 1, 8-cineole, and verbenone with the majority of the main constituents indicates that any one of these constituents would increase as the other decreases. So this relation should be considered during selection activities aimed at the improvement of these constituents. Moreover, the positive associations of endoborneol, α -terpineol, bornyl acetate, and β-caryophyllene to each other indicate the possibility of simultaneous improvement of these chemical traits (Table 2).

Table 2: Pearson’s correlation coefficient matrix for 12 main volatile constituents of the essential oil of 45 rosemary accessions.

Chemotypes of rosemary accessions

Based on the percentage of the investigated major constituents, the 45 rosemary accessions were grouped into six different chemotypes as follows:(1) α-pinene/1,8-cineole/camphor, (2) α-pinene/1,8-cineole/ verbenone, (3) α-pinene/1,8-cineole/endo-borneol, (4)1,8-cineole/ camphor/α-pinene, (5) verbenone/α-pinene/camphor and (6) camphor/1,8-cineole/verbenone. The result was consistent with Satyalet al. (2017), who classified rosemary population from different countries into a mixed chemotypes of (i) α-pinene/1,8-cineole, (ii) verbenone/α-pinene/camphor/1,8-cineole, (iii) myrcene/1,8-cineole/ camphor, (iv) 1,8-cineole/camphor/α-pinene, and (v) α-pinene/β- pinene/ camphene. Our finding was also in agreement with Jordan et al. (2011), who identified six mixed chemotypes for essential oils of rosemary populations from Murcia.

Chemotype 1 consisted of one released variety (Ros08) and 25 accessions (57.78%) from all collection regions except commercial farm. Chemotype 2 has four accessions (8.89%) including two accessions from Hadiya, one from Arssi and one released variety (Ros01). The third chemotype was made up of four accessions (8.89%) all from Harari except one released variety (Ros05). These three chemotypes were dominated by α-pinene but they differed from each other in their third major constituents. The result was comparable with previous findings that reported α-pinene as leading constituents in rosemary populations from France.

Chemotype 4 is dominated by 1, 8-cineole which has five accessions (11.1%) those from commercial farm, Wolaita and Gurage, and was in agreement with different authors who described 1, 8-cineole as a leading constituent for rosemary population from various countries. Similar findings were also stated by different writers. Chemotype 5 is dominated by verbenone and is comprised of four accessions all from North Shewa. Corresponding to this finding Napoli et al. (2010) reported verbenone as a leading constituent for rosemary populations from Sicily.

The six chemotype was represented by only two samples from Arssi (4.44%). The essential oil of the accessions in this chemotype was dominated by camphor and has a lower amount of α-pinene than all the rest accessions. Consistent with this finding, Varela et al. (2009) and Ram et al. (2011) found the prominent distribution of camphor for India and Spain rosemary essential oils, respectively. The overall finding of the current study was also in line with the result of other researchers who found α-pinene, 1, 8-cineole and camphor chemotypes for rosemary essential oils in different places (Table 3).

Table 3: Percentage of the dominant compounds and chemo types of rosemary essential oils across the 45 accessions.

This study demonstrated the presence of high chemical variability among rosemary accessions, which is mainly dependent on genetic factor than geographic location. This could be explained by the fact that the studied accessions were cultivated in the same location for more than two years prior to chemical characterization, and yet presented variability in their chemical profile. This can further supported by the observed chemical similarity between accessions collected from geographically distant locations; and chemical dissimilarity between accessions from geographically closer areas. The study also pointed out the existence of diverse accessions within collection regions, which makes it difficult to define a single chemotype based on the area of collection. Our finding at morphological and molecular level diversity analysis in the previous chapters also corroborated this and showed the existence of high within collection region diversity than between collection region. This could be resulted from presence of gene flow due to planting material exchange among growing communities. Exchange of plant materials among communities will minimize genetic diversity among populations but increase variability among individuals. This could also be the reason for the observed high within growing region diversity than between regions diversity.

Conclusions

Essential oils of forty-five rosemary accessions were analyzed using GC-MS and a total of forty-two compounds, representing95.85-98.89% of the total volatiles were identified. In general, the essential oil of the accessions were characterized by higher levels of α-pinene(5.08 - 40.62%), 1,8-cineole (8.13 - 38.48%), camphor (2.15 - 23%) and verbenone (1.83 - 20.25%), and their presence in all accessions. There were also some accessions (Ros05, Ros17, Ros18 and Ros34) that are characterized by the presence of higher levels of endo-borneol constituents in their oil (10.11 -12.56%). Correlation analysis among the major compounds showed a strong negative association of α-pinene, 1,8-cineole, and verbenone with the majority of the major compounds, while the correlation of camphor with the entire main constituent was not significant, except with α-pinene (r = -0.46***) and linalool ( r = -303*), indicating the difficulty of improving these compounds simultaneously. A positive association of endo-borneol, α -terpineol, bornyl acetate, and β-caryophyllene to each other was also noted, showing the possibility of concurrent improvement of these compositions.

Based on the percentage of the major constituents, six distinct chemotypes (α-pinene/1,8-cineole/camphor; α-pinene/1,8-cineole/ verbenone; α-pinene/1,8-cineole/endo-borneol; 1,8-cineole/camphor/ α-pinene; verbenone/α-pinene/camphor; and camphor/1,8-cineole/ verbenone) were identified for the studied accessions, demonstrating the presence of high levels of Chemotypic variability among the accessions. Overall, the study indicated the presence of wide chemical variability among the tested accessions, which was mainly dependent on the genetic background rather than the area of growth. The observed variability was higher among individual plants within collection areas than between collection areas, and was consistent with our findings at morphological and molecular level diversity analysis. The observed high chemical constituent variabilities revealed the potential of Ethiopian rosemary germplasm for wider application in different destinations that use rosemary products as raw materials.

Acknowledgments

The authors would like to acknowledge Ethiopian Institute of Agricultural Research and Agricultural Growth program II for finding the experiment. The authors also like to acknowledge Wondo Genet Agricultural Research Center for providing and facilitating the necessary facilities for the experiment.

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