PHENOLOGICAL CYCLE AND DIURNAL VARIATION EFFECTS ON THE VOLATILE OIL CHARACTERISTICS OF SAGE ( Salvia officinalis L.)

: This study was carried out to evaluate the effects of phenological and diurnal variation on volatile oil content and quality of Salvia officinalis L. (Lamiaceae) cultivated in Afyonkarahisar/Turkey. The harvesting times based on ontogeny were the pre-flowering, flowering and post-flowering stages. The harvesting took place three times a day, at 6:00 a.m., 2:00 and 8:00 p.m. The results showed that the highest volatile oil was obtained at 8:00 p.m. of the flowering stage (2.05%). In general, the best harvesting time was the flowering stage and the last hours of the day. The synthesis of volatile oil was almost the same at other phenological stages. The amount of volatile oil increased at the sunset time of the day in all studied stages. Among the identified of S. officinalis volatile oil compounds, oxygenated monoterpenes were the largest chemical group (52.8-68.6%). α-Thujone (13.0-35.8%) was the major compound of the most samples. The highest and lowest values of this compound were observed in the post-flowering and flowering stages, respectively. The other main compounds were camphor (7.0-20.2%), 1,8-cineole (6.9-14.1%), borneol (2.8-15.8%) and veridiflorol (4.5-12.3%). The effects of climatic factors such as day length, insolation, temperature and plant growth stage affected the quantity and quality of volatile oil content of S. officinalis . The results showed that the best harvesting time for S. officinalis for volatile oil content is 8:00 p.m. at the flowering stage in Afyonkarahisar climatic conditions. The volatile oil compositions of the plant varied widely at different harvest times (ontogeny and diurnal)

The production of secondary metabolites in plants as bioactive chemicals is a natural response to biotic and abiotic stresses that improve plant resistance to adverse conditions (Ramakrishna & Ravishankar 2011).The biosynthesis of secondary metabolites in terms of quality and quantity, although controlled genetically, is strongly affected by the climatic conditions, environment organisms, applied agro-techniques, harvesting time and post-harvesting processing (Miguel et al. 2004, Soltanbeigi & Sakartepe 2020).In other words, the volatile oil synthesis depends on the interaction between genetic, ontogenesis and physiological state of the plant with environmental conditions.The regulation of the volatile compounds is further complicated by dynamic differential components of abiotic factors such as edaphic properties, moisture, temperature and light intensity (Lee & Ding 2016).Ontogeny acts as a timeline for plant growth, which has a major role in determining the maximum accumulation time of volatile oils (Verma et al. 2012).The accumulation pattern of secondary metabolites in plants is characterized by diurnal fluctuation due to enzymatic activities, temperature and light (Asghari et al. 2014).Knowledge of factors that increase the quality and quantity in agro-ecosystems is necessary.These factors largely depend on the type of plant that can be considered to achieve cultivation aim.Therefore, methods that can produce a healthy medicinal plant with more effective substances may be needed (Azarnivand et al. 2010).Since climatic conditions cannot be controlled and managed, planting time, agronomic practices and especially harvesting time are the most critical factors in the optimal production of volatile oil and its quality (Lakušić et al. 2013, Katar et al. 2019).
This study aimed to determine the appropriate harvest time for S. officinalis in terms of ontogenetic and diurnal variability in Afyonkarahisar climate conditions.

Plant Material, Location
The plant material was 3-year-old Salvia officinalis.The samples were obtained from an ongoing study in Afyonkarahisar Medicinal and Aromatic Plants Centre (38° 46' N, 30° 30' E) located in Turkey's inner Aegean region.The region is characterized as a transitional climate zone affected by three main climatic conditions seen in the Mediterranean (South), Central Anatolia (East) and Aegean (West) regions.The climate of the experimental location is harsh, with moderate rainy.Most precipitation occurs in winter and spring.Summers are hot and dry and winters are cold and snowy.Some meteorological data during the sampling season are given in Table 1 and some physico-chemical properties of the soil of the sampling area are given in Table 2.

Treatments
Plants allocated for sampling were grown following sustainable farming methods.Samples were taken at three different growth and developmental stages (ontogenetic variability) and three different hours of the day (diurnal variability).The pre-flowering (1 June), full flowering (24 July) and post-flowering (29 August) stages were chosen for ontogenetic variability samples.The hours for diurnal diversity were 6:00 a.m., 2:00 p.m. and 8:00 p.m.The plant samples were cut from 5-8 cm and were separated to leaves and stems.The leaves were dried at 37°C in a cabinet type dryer for 72 hours.

Volatile oil isolation
For VO isolation, 50 grams of dry leaves were subjected to hydro-distillation with distilled water (1:10) by a neo-Clevenger type apparatus for three hours in 3 replications.The VO content was determined volumetrically (mL 100 gr -1 ).After decantation, the VO samples were dried over anhydrous sodium sulfate and kept in amber vials at -4°C.

Identification of chemical compounds
The chromatographic analysis was conducted by a gas chromatography (GC) system (Agilent 7890B-USA) equipped with a flame ionization detector (FID) and coupled to a mass spectrometry detector (MSD) (Agilent 5977A-USA) under electron impact ionization (70 eV).The MS scan range was 50-550 atomic mass units (AMU).The column for the separation of the compounds was HP-Innowax (Agilent 19091N-116: 60 m × 0.320 mm internal diameter and 0.25 μm film thickness).The carrier gas was helium at a flow rate of 1.3 mL min -1 .The detector, injector and ion source temperatures were 270°C, 250°C and 230°C, respectively.Injection volume was set at 1 μL (20 μL VO diluted in 1 mL n-Hexane) in split mode (40:1).The samples were analyzed with the column held initially at 70°C for 5 min hold time.Then, the temperature was raised to 160°C with a 3°C min -1 heating ramp and 5 min hold time.Eventually, the temperature reached to 250°C with a 6°C min -1 heating ramp and 5 min hold time.For accurate determination of chemical compounds, the retention indices (RI) were calculated by injecting C7-C30 n-alkanes (Sigma-Aldrich-USA) to (GC/FID) system (Agilent 7890B-USA) under the same conditions of the VO analyses.The identifications of the VO components were performed by comparing retention indices, mass spectra by the computer library database of US National Institute of Standards and Technology (NIST), Wiley libraries, other published mass spectra data (Adams 2007) and our database.Relative abundance (% area) was calculated based on the ratio between each compound's peak area and the sum of areas of all compounds.No response factors were calculated.

Results and Discussions
The distillation step revealed that the samples' VO content varied by different growth stages and different hours of the day (1.0-2.05mL 100gr -1 ).In terms of ontogenetic variability, the maximum VO contents was obtained from samples taken in the full flowering stage (1.4-2.05mL 100gr -1 ).The VO content VO increased diurnally in all three stages of the harvests as hours progressed during the day (1.3-2.05mL 100gr -1 ) (Table 3).Although genetics is considered to be the crucial determinant of quantity and quality (  A total of 44 components were obtained as a result of GC-MS analyses (Table 3).The highest number of components was found at 2:00 p.m. sample related to the pre-flowering stage.The least component was obtained at 2:00 p.m. in the full flowering stage.Some components were not found in different growth stages and different times of the day.For instance, tricyclene, sabinene and 1octen-3-ol were not found in the pre-flowering samples, sabinyl acetate, alloaromadendrene and (+)-spathulenol were not detected in the pre-flowering and flowering stages, cis-Ocimene, α-copaene, linalool and propanal, 2methyl-3-phenyl-did not appear in the flowering and post-flowering stages.Aromadendrene and γ-cadinene were not identified in the flowering and post-flowering stages, respectively.Oxygenated monoterpenes were the main chemical group at different phenological stages with a considerable difference (52.8-68.6%)(Table 3).The highest accumulation of oxygenated monoterpenes occurred in the post-flowering stage, followed by flowering and preflowering stages, respectively.In other words, with the aging of the plants, the amounts of oxygenated monoterpenes increased.Although the highest level of oxygenated monoterpenes was observed at 8:00 p.m. of the pre-flowering stage, this group's amounts were higher in the morning harvests (6:00 a.m.) of flowering and postflowering stages.This group was followed by hydrocarbon sesquiterpenes (6.6-19.6%),hydrocarbon monoterpenes (4.7-14.3%),oxygenated sesquiterpenes (7.0-13.6%),oxygenated diterpenes (2.1-6.5%) and esters (0.6-4.5%).The highest level of hydrocarbon monoterpenes was observed in the flowering and postflowering stages.This group lacked specific behavior in terms of diurnal changes.However, the total amount of monoterpenes (oxygenated monoterpenes + hydrocarbon sesquiterpenes) in VO was highest at 6:00 a.m.Levels of both oxygenated sesquiterpenes and sesquiterpene hydrocarbons decreased with the aging of the plants.This state was in contrast to monoterpene's behavior.Epimanool, as a labdane diterpenoid, was the only oxygenated diterpene in VO samples of S. officinalis.The highest amounts of this composition were identified in the pre-flowering stage.As the hour progresses during the day, the values of this compound increased in all three phenological stages.Also, the highest accumulation of esters was in the flowering stage (Table 3).According to Piccaglia et al. (1997) 2013) on S. officinalis, the total monoterpene compounds increased in the warm months of the year and at the same time, the sesquiterpene compounds decreased, as in the case of our findings.The amount of monoterpene hydrocarbons at the flowering stage was also reported to be higher than at other phenological stages (Mirjalili et al. 2006), similar to our findings, but the sum of monoterpenes was higher in the flowering stage.Rguez et al. (2018) indicated that the amount of oxygenated monoterpenes in the late afternoon hours of the day (5:00 p.m.) was at the highest level and monoterpene hydrocarbons were at the lowest level.But the sum of these two groups increased as the hour progresses during the day.
Except for the samples taken at 2:00 p.m. and 8:00 p.m. in the flowering stage, α-thujone (13.0-35.8%)was identified as the major compound of S. officinalis VO.The lowest (13.0%) and highest (35.8%) content of αthujone was found at 8:00 p.m. in the flowering stage and 2:00 p.m. in the post-flowering samples, respectively.In terms of ontogenetic variation, the highest α-thujone content was in the after-flowering stage (32.3-38.8%).The pre-flowering and flowering stages followed the postflowering stage.The highest α-thujone in the preflowering and flowering stages were recorded at 6:00 a.m.(23.8 and 19.2%) (Table 3).According to observations of Mirjalili et al. (2006) and Ben-Farhat et al. (2016), the maximum amount of α-thujone was in the post-flowering stage (fruiting stage) and the lowest level was in the flowering stage.Katar et al. (2019) also reported the highest amounts of this compound in the full-flowering and fruiting-set stages.As the aging of S. officinalis, α-thujone biosynthesis accelerates, which almost coincides with the warmer months of the year (Lakušić et al. 2013).A study that examined diurnal variations in S. officinalis VO compositions, α-thujone showed no clear response to hour changes (Rguez et al. 2018).
The other main constituents identified were camphor (7.0-20.2%),1,8-cineole (6.9-14.1%),borneol (2.8-15.8%)and veridiflorol (4.5-12.3%).The highest camphor content was found at 2:00 p.m. sample in the flowering stage (20.2%), and the lowest content was seen at 6:00 a.m. of pre-flowering.Although this compound's highest and lowest values were observed at 2:00 p.m. and 6:00 a.m.samples in the flowering and pre-flowering stages, this was not the case in the post-flowering stage.According to α-thujone and camphor action, it was observed that with the raising of α-thujone content, the content of camphor decreased in each growth stage.The highest content of 1,8-cineole (14.1%) was found in the pre-flowering stage.The highest values of this component were detected at 6:00 a.m.samples in all three growth stages.Besides, as the plants got older, 1,8-cineole biosynthesis was adversely affected.The greatest formation of borneol occurred during the flowering stages (10.2-15.8%).An increase was observed in the content of this compound from early morning hours to sunset.The highest and lowest contents of veridiflorol were identified at 2:00 p.m. sample of pre-flowering and the 6:00 a.m.sample of post-flowering, respectively.Veridiflorol contents decreased with plant aging.The other important compounds of S. officinalis VO were α-pinene (0.6-4.2%), camphene (1.0-5.2%),β-pinene (0.7-2.6%), βthujone (1.1-4.9%),caryophyllene (2.1-8.6%),αhumulene (3.3-10.9%),and epimanool (2.1-6.5%).α-Pinene and camphene had higher contents in the postflowering and flowering stages, respectively.β-thujone was found in minimal quantities during the flowering stage.The highest contents for caryophyllene, αhumulene and epimanool were seen in the post-flowering stage.However, these components do not have clear action in terms of diurnal variability.
The amounts of the major constituents of current findings such as α-thujone (13-35.8%),camphor (7-20.2%)and 1,8-cineole (6.9-14.15%)were largely within the range of the proposed standards.Most of the compounds found in the present study have been reported in other studies on S. officinalis with different amounts (Mirjalili et al. 2006, Ben-Farhat et al. 2016, Rguez et al. 2018, Katar et al. 2019).

Conclusion
In this study, the influence of phenological cycle and diurnal variation were investigated on the content and chemical properties of VOs of S. officinalis sampled in Afyonkarahisar/Turkey.The quantity and quality of plant VOs were significantly affected by harvesting times.Based on the obtained results, the highest synthesis of VO occurred in the full flowering stage and at 8:00 p.m.The VO compositions of the plant varied widely at different growing stages and harvesting times (ontogeny and diurnal).The effects of climatic factors such as day length, insolation, temperature and also plant growth stage on these changes were evident.Knowing the agronomic and chemical properties and accurately determining the biologically active compounds of medicinal plants at different growth stages provide a clear perspective for their purposeful use including cosmetic, medicine and food industries.Extensive research is being conducted on adaptation of S. officinalis, including the effects of optimal planting time, density, nutrition and irrigation on the quantity and quality of the plant under the climatic conditions of Afyonkarahisar.
low light and low temperatures at harvest time are likely to cause internal conversions between monoterpenes and sesquiterpenes.Increased oxygenated monoterpenes of S. officinalis VO in the postflowering stage (fruiting) have been reported by Ben-Farhat et al. (2016) and Mirjalili et al. (2006).In the study of Lakušić et al. (

Table 1 .
Some local meteorological data of the experimental field in 2019.

Table 2 .
The values of some physico-chemical properties of the experimental soil.

Table 1
Lee & Ding 2016), it is inferred that in addition to agricultural practices, plant age, day length, light intensity, day and night temperature changes and relative humidity affect the VO content (Soltanbeigi 2020).Based on European Pharmacopoeia (2005) standards, the minimum VO content of S. officinalis should not be less than 1.5% in dried leaves.According to Paulus et al. (2019), the sunlight (quality, intensity and duration) has the most effect on VO synthesis.The flowering stage in this study coincided with July, when the plants received most of the light intensity ( ). Ben-Farhat et al. (2016) showed that the best time to harvest S. officinalis is the flowering stage (1.49%) in terms of VO content.Besides, the process of biosynthesis of VOs in plants takes place only in very young cells.Since the accumulation of VO is directly related to leaf ontogeny, its synthesis rate is rapid in the early stages of leaf growth.With increasing leaf weight, its VO content remains almost constant (Nurzyńska-Wierdak et al. 2012).Ramezani et al. (2009) indicated the temperature variation during the day has more effect on VO accumulation.More recently, Rguez et al. (2018) reported an increase in S. officinalis VO content from morning to afternoon (7 a.m.: 0.78%, 12 noon: 0.93% and 5 p.m.: 1.1%).

Table 3 .
The content and chemical characteristics of Salvia officinalis volatile oil affected by phenological cycle and diurnal variation.