Anatolian sage clones' mean essential oil content and main components of 1,8-cineol, camphor, β-pinene, β-caryophyllene and β-thujone were 3.09 50.21, 11.29, 8.92, 5.74 and 1.87, respectively (Table 3). The values were compatible with those reported in other studies (Skoula et al. 2000; Leontaritou et al. 2020; Karık and Sağlam, 2018).
Table 3
Essential oil ratio and mean, minimum, maximum values and standard deviations of essential oil main components
Parameter/Traits | Essential oil | 1,8 Cineol | Camphor | β-Pinene | β-Caryophyllene | β-Thujone |
Mean | 3.09 ± 0.04 | 50.21 ± 0.3 | 11.29 ± 0.4 | 8.92 ± 0.1 | 5.74 ± 0.2 | 1.87 ± 0.06 |
Minimum | 1.67 | 34.69 | 0.44 | 3.66 | 0.55 | 0.32 |
Maximum | 6.00 | 67.85 | 33.29 | 18.40 | 15.68 | 8.18 |
Standard deviation | 0.76 | 5.81 | 7.51 | 2.77 | 2.77 | 1.24 |
Coefficient of variation (%) | 24.58 | 11.56 | 66.51 | 31.04 | 52.74 | 66.09 |
Table 4
Variance analysis for the traits
Traits | Source of Variation | Degrees of Freedom | Mean Squares | F Value | P Value |
Essential oil | Clone | 6 | 0.21 | 0.74 | 0.626 |
Harvest Time | 11 | 16.67 | 168.22 | < .0001 |
Clone*Harvest time | 66 | 0.14 | 1.63 | 0.0030 |
Year | 1 | 16.54 | 166.89 | < .0001 |
Block | 2 | 0.17 | 0.66 | 0.5331 |
Clone*Block | 12 | 0.28 | 2.83 | 0.001 |
Error | 392 | 0.10 | | |
1,8-cineol | Clone | 6 | 540.04 | 76.87 | < .0001 |
Harvest Time | 11 | 430.97 | 28.74 | < .0001 |
Clone*Harvest time | 66 | 27.74 | 2.23 | < .0001 |
Year | 1 | 500.86 | 33.40 | < .0001 |
Block | 2 | 16.59 | 2.20 | 0.1416 |
Clone*Block | 12 | 6.89 | 0.46 | 0.9372 |
Error | 392 | 15.00 | | |
Camphor | Clone | 6 | 2.70 | 28.07 | < .0001 |
Harvest Time | 11 | 42.10 | 117.74 | < .0001 |
Clone*Harvest time | 66 | 0.730930 | 2.59 | < .0001 |
Year | 1 | 2.22 | 6.22 | 0.013 |
Block | 2 | 0.18 | 1.64 | 0.2176 |
Clone*Block | 12 | 0.90 | 0.26 | 0.9947 |
Error | 392 | 0.36 | | |
β-pinene | Clone | 6 | 157.54 | 130.05 | < .0001 |
Harvest Time | 11 | 115.02 | 44.14 | < .0001 |
Clone*Harvest time | 66 | 7.26 | 4.37 | < .0001 |
Year | 1 | 20.08 | 7.71 | 0.0058 |
Block | 2 | 0.67 | 0.52 | 0.6061 |
Clone*Block | 12 | 1.19 | 0.46 | 0.9391 |
Error | 392 | 2.60 | | |
β-caryophyllene | Clone | 6 | 2.16 | 11.07 | 0.0003 |
Harvest Time | 11 | 10.24 | 103.93 | < .0001 |
Clone*Harvest time | 66 | 0.13 | 1.37 | 0.0416 |
Year | 1 | 17.88 | 181.50 | < .0001 |
Block | 2 | 0.10 | 2.00 | 0.6144 |
Clone*Block | 12 | 0.20 | 2.00 | 0.0234 |
Error | 392 | 0.10 | | |
β-thujone | Clone | 6 | 6.67 | 64.11 | < .0001 |
Harvest Time | 11 | 0.55 | 10.08 | < .0001 |
Clone*Harvest time | 66 | 0.10 | 2.23 | < .0001 |
Year | 1 | 0.16 | 2.95 | 0.0864 |
Block | 2 | 0.09 | 0.95 | 0.413 |
Clone*Block | 12 | 0.10 | 1.93 | 0.0291 |
Error | 392 | 0.05 | | |
The results of variance analysis are given in Table 4. The differences between clones, harvest times, clone harvest x time interaction and years (except β-thujone) were statistically significant, except essential oil content. The essential oil content and essential oil components of medicinal and aromatic plants may vary depending on ecological conditions, climate and soil characteristics, genotypic structure, part of the plant used and harvest time (Saharkhix et al. 2009). The significant difference (P < 0.01) in the traits (except essential oil) among the clones shows the importance of genetic structure (Table 4). The mean values of traits for the clones are given in Table 5. The difference between the lowest and the highest ratio in the β-pinene component was nearly two-fold, while the difference between these values was nearly 4-fold for β-thujone. The highest 1,8-cineol, one of the main components in essential oil, content (56.53%) was recorded in fd4-13, followed by fd2-9. The lowest β-thujone ratio (0.99%) was obtained from fk3-16, while the highest ratio (3.57%) was obtained from Bk4-9. The clones with the highest component in each trait varied, while the fd4-13 clone had the highest 1.8-cineol and β-pinene ratio. The results revealed that the clonal selection is successful, and the differences are still maintained in the clones obtained at the end of the selection.
Table 5
Mean (%), standard errors and overall mean of essential oil content, and essential oil main components of genotypes (no beta thujone)
Clone | Essential oil | 1,8 Cineol | Camphor | β-Pinene | β-Caryophyllene | β-Thujone |
fd4-13 | 3.00 ± 0.10 | 56.53 ± 0.96 | 8.28 ± 0.21 | 10.26 ± 0.31 | 4.20 ± 0.08 | 1.61 ± 0.03 |
fd2-9 | 3.09 ± 0.09 | 51.96 ± 0.67 | 8.71 ± 0.17 | 10.36 ± 0.33 | 4.57 ± 0.75 | 1.03 ± 0.02 |
fk3-16 | 3.08 ± 0.09 | 50.77 ± 0.78 | 8.99 ± 0.18 | 10.29 ± 0.35 | 4.84 ± 0.07 | 0.99 ± 0.02 |
fk4-14 | 3.16 ± 0.10 | 50.71 ± 0.73 | 9.66 ± 0.16 | 9.13 ± 0.36 | 5.25 ± 0.07 | 1.46 ± 0.03 |
standard | 3.06 ± 0.09 | 49.15 ± 0.46 | 12.02 ± 0.11 | 7.29 ± 0.17 | 5.46 ± 0.09 | 1.81 ± 0.03 |
Fk4-9 | 3.15 ± 0.09 | 48.00 ± 0.40 | 9.94 ± 0.10 | 8.45 ± 0.20 | 6.14 ± 0.07 | 3.57 ± 0.05 |
fk5-7 | 2.97 ± 0.12 | 44.94 ± 0.54 | 11.42 ± 0.15 | 5.68 ± 0.21 | 7.25 ± 0.11 | 2.34 ± 0.03 |
Overall Mean | 3.07 | 50.29 | 9.86 | 8.78 | 5.39 | 1.83 |
Many researchers carried out studies on several aspects of Anatolian sage (Bayram et al. 1999, Baydar et al. 1999, Skoula et al. 2000, Aydın et al. 2019, Dinçer et al. 2012, Karık and Sağlam 2017, Karık and Sağlam 2018, Leontaritou et al., 2020). The essential oil ratios and main components obtained from 6 clones in the current study are similar to the previous studies. However, previous studies were carried out using Anatolian sage produced from seeds, while this study was carried out using the clones. Clonal studies are important to show the superiority of clones to obtain sustainable products at desired quality and quantity compared to the individuals produced from seeds. In addition, the findings obtained from the clones also indicated that clones could be selected according to the desired component.
The essential oil ratio at the harvest times statistically differed for all of the clones (Table 4), and the mean essential oil ratios of the different clones are given in Table 6. The highest essential oil ratio (3.93%) was obtained in August, while the lowest oil ratio (2.10%) was recorded in February. The highest percentage of β-thujone (2.12%) was obtained in October and the lowest (1.05%) was in March. The ratios of other components also significantly varied with the harvest time. The relationship between harvest time and essential oil content was associated with temperature and light intensity, and the decrease in temperature and light intensity caused a decrease in the essential oil ratio (Kargiolaki et al. 1994). This study showed that essential oil and camphor reached the highest values in August when light and temperatures were higher. The results are consistent with the findings of Çiçek et al (2011), who obtained the highest essential oil content (4.58%) in Salvia officinalis in August. Therefore, the plants should be harvested in August to produce essential oil and camphor.
Determining the highest essential oil ratio for medicinal plants is not sufficient; however, the variation of main components of essential oil with the harvest time also is of great importance. Therefore, the present study determined the effect of harvest time on essential oil components. The results indicated that the ratio of some components significantly differed with the harvest time.
Table 6
Monthly variation of essential oil contents and main components of essential oil
| Essential oil | 1,8-cineol | Camphor | β-pinene | β-caryophyllene | β-thujone |
January | 2.36 h | 51.22 c | 5.31 f | 10.18 b | 7.95 a | 1.89 ab |
February | 2.10 i | 51.74 bc | 4.02 f | 10.60 b | 8.09 a | 1.68 ab |
March | 2.11 i | 52.65 abc | 2.44 g | 12.33 a | 7.88 a | 1.05 c |
April | 2.44 h | 53.80 ab | 4.05 f | 10.53 b | 7.75 a | 1.25 c |
May | 3.72 bc | 54.71 a | 10.37 d | 7.68 d | 5.15 c | 1.68 ab |
June | 3.37 ef | 52.69 abc | 13.21 c | 7.69 d | 4.37 d | 1.63 b |
July | 3.60 cd | 48.48 d | 18.55 ab | 6.85 d | 2.45 f | 1.94 ab |
August | 3.93 a | 44.05 e | 21.36 a | 6.90 d | 2.46 f | 1.81 ab |
September | 3.82 ab | 44.78 e | 19.68 ab | 7.29 d | 2.66 f | 1.96 ab |
October | 3.48 de | 46.86 d | 17.55 b | 7.68 d | 3.65 f | 2.12 a |
November | 3.30 f | 51.06 c | 8.64 e | 9.26 c | 6.28 b | 1.89 ab |
December | 2.80 g | 50.94 c | 5.60 f | 10.96 bc | 8.20 a | 1.80 ab |
The lowest and highest ratios of the camphor are approximately nine times different (Table 6). The β-binene, one of the important main components, reached its highest level (12.33%) in March, while it decreased by 1.7 times in July and reached its lowest level. İn contrary, β-caryophyllene reached the highest level (8.20%) in December, whereas it decreased by 3.4 times in July to 2.45%. the 1,8-cineol ratio was the lowest (44.05%) in August, when the essential oil was high, on the contrary, camphor, which was stated to have a toxic effect by Narayan and Singh (2012), had the highest ratio (21.04%) in August. Similarly, Sarrou et al. (2016) found that essential oil was the highest (5.60%) in the summer, while the ratio of 1.8 cineoles was the lowest (44.70%). On the other hand, β-thujone, which is among the unwanted components in some sectors (Hold et al. 2000), was the lowest in March (Table 6). In addition, Hold et al. (2000) reported the highest values for camphor (14.93%) and β-thujone (2.79%) in summer. There are seasonal similarities between the findings of Hold et al (2000) and the current study, while the results are slightly different monthly. The differences may be attributed to rain or harvest or collection time temperatures. Similarly, the ratio of α-pinene and β-caryophyllene harvested in April and May in Greece was reported as 4.19 and 9.74%, respectively (Leontaritou et al. 2020). Sarrou et al. (2016) conducted a study between April and October and found the highest ratio of β-pinene (14.07%) and β-caryophyllene (7.17%) in April. The differences in findings suggest that the harvest time is an important factor. In addition to harvest time, Sarrou et al. (2016) stated that water is influences essential oil yield and components, while Kargiolaki et al. (1994) stated that temperature and light were effective. The content and composition of essential oil in the plants depend on the plant parts collected or harvested (Bellomaria et al., 1992), different development stages of the plant (Porres-Martínez et al. 2014, Kara, 2020), post-harvest drying temperature (Venskutonis, 1997, Aydın et al. 2019) and storage conditions (Dinçer et al. 2012).
The ratio of essential oil components for each clone significantly differed with the months (clone harvest time interaction) (Annex 1). The relevant tables are presented as an appendix since all tables will occupy too much space in the text. The highest 1,8-cineol ratio (63.80%) was detected in fd4-13 samples harvested in March, followed by the Fd2-9 clone harvested in April (58.52%). The lowest 1.8 cineol ratio (39.77%) was obtained in fk3-16 harvested in August. Although the 1,8-cineol ratio differed with the clones, the highest ratio was obtained in plants harvested in March, April, May, and June (Annex 2). While the lowest camphor rate (0.92%) was obtained from fk3-16 clone in March, the highest camphor rate was detected in the same clone in August (Annex 3). According to the months, the β-pinene content among the clones varied between 4.75% and 14.95%. In general, β-pinene was detected at a high rate in the spring months, while it was highest in January in the fd4-13 clone (Annex 4). According to the harvest time of the clones, the highest content of β-caryophyllene (12.18%) was determined in the fk5-7 clone in April (Annex 5). The results revealed that the component ratios of each cultivar and/or cultivar candidate developed may differ according to the harvest time. Different harvesting period for each of clones enables to obtained higher yields.
A correlation test was also carried out between the ratio and essential oil components. The highest negative correlation (-0.80) was recorded between camphor and β-binene, and followed by the correlation between essential oil and β-caryophyllene (-0.72). The β-pinene and β-caryophyllene, which are essential oil components, have various biomedical properties. Leite et al. (2007) demonstrated the antibacterial effect of α and β-pinene components. In addition, Astani and Schnitzler (2014) reported that β-pinene has an antiviral effect and reduces viral infectivity by 100%. Koyama et al. (2019) determined the flavoring and wound healing properties of β-caryophyllene. The findings showed that camphor increases while β-pinene decreases, and β- caryophyllene decreases while essential oil increases. The results revealed that sage clones could be determined according to the production of targeted components, and the essential oil yield and the ratio of components can be increased together.
Table 7
Correlation between essential oil ratio and essential oil main components
Trait | Essential oil | 1,8-cineol | Camphor | β-pinene | β-caryophyllene | β-thujone |
Essential oil | 1 | | | | | |
1,8-cineol | -0.23 | 1 | | | | |
< .0001 | | | | |
Camphor | 0.69 | -0.62 | 1 | | | |
< .0001 | < .0001 | | | |
β-pinene | -0.50 | 0.54 | -0.80 | 1 | | |
< .0001 | < .0001 | < .0001 | | |
β-caryophyllene | -0.72 | 0.06 | -0.67 | 0.34 | 1 | |
< .0001 | 0.193 | < .0001 | < .0001 | |
β-thujone | 0.20 | -0.29 | 0.26 | -0.47 | -0.02 | 1 |
< .0001 | < .0001 | < .0001 | < .0001 | 0.6801 |
The components in the essential oil can be beneficial, while they can also have many toxic effects depending on the dose. Many studies have been carried out on the toxicology of the thujone component. Lachenmeier et al. (2006) stated that thujone acts on the central nervous system, while Olsen (2000) reported that thujone is toxic but has a stimulating effect on the brain instead of affecting the central nervous system. In another study, Nikolić et al. (2015) revealed that camphor has a mutagenic effect. In addition, Narayan and Singh (2012) reported that camphor may have a toxic effect, especially on young children, while Chen et al. (2013) reported that camphor could cause miscarriages. Therefore, production planning should be carried out considering the correlations determined in our study for the compounds as mentioned earlier.
The results revealed that the clones with the desired components could be selected among six by considering essential oil and components. In case of production for 1.8 cineol, the fd4-13 clone can be selected (Table 5), and this clone should be harvested in March to obtain the highest 1.8 cineol ratio (Annex 2). The negative correlation (-0.62) between 1.8 cineol and camphor in March harvested plants indicates that the camphor ratio will be low, in contrast, the positive correlation (0.54) between 1.8 cineol and β-pinene indicates that β-pinene will be at a moderate level (Table 7).
The essential oil component ratios of clones at the harvest time are taken into account for the selection of Anatolian sage clones, which are used for many purposes in different sectors. Selecting the clones only for their essential oil ratio at harvest or collection time may cause problems in terms of the presence of some toxic substances in the essential oil and also the desired components in the essential oil may be lower at harvest time. The difference in the content and components of essential oil between clones reveals the importance of the standard raw material for the standard product. The standard raw materials can be obtained by developing suitable varieties on a sectoral basis. The current study reveals different options in this respect. For example, Valussi et al. (2021) stated that a camphor ratio higher than 10–23% might cause mucosal irritation and gastrointestinal problems. The fd4-13 clone with low camphor content can be recommended for the cosmetic industry. In addition, for thujone, which is stated to have a toxic effect above a certain dose Zeybek and Zeybek (1994) and Hold et al. (2000), the fd2-9 and fk3-16 clones with trace amounts of thujone can be recommended for the tea industry. Major component 1,8 cineol has antitussive (Fischer and Dethlefsen, 2013), mucolytic (Juergens et al., 2020), anti-inflammatory (Juergens et al., 2003) and antimicrobial (Juergens et al., 2020) characteristics (Valussi et al. 2021). In this context, the fd4-13 clone can be recommended. The fd2-9 clone with high 1,8-cineol and low thujone has market demanded essential oil component ratios.
The results revealed that the component ratios vary according to the clones, the component contents of clones differ according to the harvest time, and there are significant correlations between the components. In addition, the clones were rich in some components and low in some toxic components. The correlations between components on a seasonal basis have been ignored in many breeding studies. The correlations between harvest time and essential oil components of clones offer the opportunity to evaluate different options for producers and consumers. Proper use of options by the producers will contribute to more sustainable production and consumption of sage components.