Evaluation of harvesting time and standardization of distillation duration for higher essential oil content and quality in German chamomile (Chamomilla recutita L.)

Essential oil yield and composition in aromatic crops might be affected by genetic, agronomical and environmental factors but till date there is no clear information about the harvesting time and distillation for higher essential oil content without affecting quality. The current study was carried out to evaluate harvesting of chamomile flowers without herb and with herb part at three different times (6 A.M., 12 P.M. and 6 P.M.) and four distillation treatments (3 h, 4 h, 5 h & 6 h) for dried chamomile flowers. Results indicated that essential oil content was more in chamomile flowers without herb (0.15-0.18%) as compared to flowers with herb (0.06-0.09%). Essential oil content in chamomile flowers without herb was found statistically at par at harvest time of 12 P.M. (0.18%) and 6 P.M. (0.18%) and significantly higher than harvesting time of 6 A.M. (0.15%). Essential oil of chamomile flowers without herb contained maximum α-bisabolol oxide-B, (Z)-spiroether, and chamazulene at 12 P.M. and 6 P.M. while, α-bisabolone oxide-A and α-bisabolol oxide-A were maximum at 6 A.M. and (E)-β-farnesene was more at 12 P.M. Similarly, in distillation experiment, higher oil content was observed in chamomile dried flowers which were hydro-distilled for 6 h (1.20%) compared to other hydro-distillation durations. Marker compounds i.e. α-bisabolol oxide-A, α-bisabolone oxide-A, α-bisabolol oxide-B, (E)-β-farnesene and chamazulene were more at 5 h and 6 h distillation duration while (Z)-spiroether was more at 3 h distillation duration. The present study showed that in order to obtain higher essential oil, flowers without herb harvested at 12 P.M or 6 P.M. should be subjected to 5-6 h hydro-distillation.


Introduction
Chamomile (Chamomilla recutita L.) is one of the oldest and very well known medicinal herb which belongs to Asteraceae family. C. recutita is also known as German chamomile. Botanically, it is annual herb with widespread roots. Its height varies from 40 to 70 cm, depending on the location, climate and 141 soil type. Chamomile is a native crop to Iran and Europe where it grows as a wild plant (Reichinger 1977;Pourohit & Vyas 2004). Chamomile is very well known for its medicinal properties like antiinflammatory, anti-spasmodic, anti-bacterial and anti-septic etc. (Franke & Schilcher 2007). Chamomile contains about 0.24-1.9% essential oil in which more than 120 compounds have been identified (Kumar et al. 2016). Chamomile essential oil is also known as blue oil in national and international markets. Marker compounds of chamomile oil are bisabolol oxides, farnesene, α-bisabolone oxide-A, α-bisabolol and chamazulene which are actively involved in reducing inflammation and other medicinal properties (Jakolev et al. 1983). Nationally and internationally, presence of marker compound in chamomile essential oil makes it a commodity of great demand which is increasing day-by-day besides its medicinal properties. Chamazulene, one of the most important component responsible for blue coloration of chamomile essential oil was derived from matricine by the process of pH changes during distillation process . Essential oil content and its composition vary due to genetic variability or environmental factors. Single or a combination of environmental factors influence the content of essential oil and also its composition. In general, temperature and light fluctuation throughout the day affect the essential oil content and composition in aromatic crops Asghari et al. 2014). In chamomile, harvesting of flowers is cumbersome which increases the production cost. According to Salamon (2007), maximum chamomile flowers production may be achieved by efficient and effective collection of flowers at right time and stage. It is also essential to know how essential oil content and composition changes during daytime and seasons. Seasonal variations of chamomile essential oil yield have been previously studied by several researchers (Pino et al. 2002). Diurnal variation of chamomile essential oil has not been extensively studied yet.
Besides genetic, environmental and agri-practices, distillation duration during essential oil extraction also plays an important role in yield and composition of essential oils. Till date, no literature is available on optimization of distillation duration for essential oil extraction to increase yield. Hence, the objectives of the present study were to find the most appropriate harvesting time of chamomile flowers for higher essential oil content without affecting quality and also to standardize the distillation duration to get maximum essential oil content.

Plant sample
Plant material for experiment was collected from chamomile plant variety CIM-Sammohak available at the Experimental Farm of CSIR-Central Institute of Medicinal & Aromatic Plants, Research Centre, Pantnagar. Harvesting of chamomile flowers was done in full blooming stage at three times during the day (6 A.M, 12 P.M and 6 P.M). Required amount of fresh flowers were harvested in two ways i.e. flowers with herb part and flowers without herb part on the same day and for optimization of harvesting time and plant part with respect to essential oil content.

Essential oil extraction
Samples were subjected to hydro-distillation just after harvesting on the same day in March, 2020. Rest of the samples were air dried at room temperature (<25 0 C) for 10-15 days to determine essential oil content in dried chamomile flowers. As per experimental design, the extracted oil from samples were used for optimising the distillation duration for maximum recovery of essential oils, the dried flowers were hydro-distilled for 3 h, 4 h, 5 h and 6 h by using glass Clevenger apparatus to produce oil by recommended method. The oil volume was measured directly in the extraction burette. The essential oil content (%, v/w) was calculated as volume of oil (mL) per 100 g of fresh and dried plant material. The obtained oil was dried over anhydrous sodium sulphate in sealed vial at low temperature.

GC-MS analysis
The essential oil samples were analysed by GC-FID and GC-MS techniques. GC-FID analysis of essential oil was carried out on Thermo Fisher Trace GC-1300 Essential oil content and quality of German chamomile at varied harvesting and distillation time equipped with TG-5 (30 m × 0.25 mm; 0.25 µm film thickness) fused silica capillary column and with flame ionization detector (FID). Nitrogen was used as carrier gas at a flow rate of 1.0 mL min -1 . The oven temperature programming was done from 60-230°C with an increase of 3°C min -1 . The injector and detector temperatures were 230°C and 250°C, respectively. The injection volume was 0.02 µL neat with a split ratio of 1:40. Gas chromatography-mass spectrometry (GC-MS) analysis of essential oil was performed on Perkin-Elmer Turbomass Quadrupole Mass Spectrophotometer fitted with PE-5 (Perkin Elmer) fused silica capillary column (30 m × 0.25 mm; film thickness 0.25 µm). The oven column temperature was programmed from 60 to 240°C with an increase of 3°C min -1 with initial and final hold time of 2 min, using helium as carrier gas at a flow rate of 1.0 mL min -1 . The injector and ion source temperatures were maintained constant at 250°C. The injection volume was 0.02 µL neat with split ratio 1:30. MS were taken at 70 eV (EI). The essential oil constituents were identified by comparing retention indices (RI), mass spectra library search and by comparing their mass spectra (MS) data with literature data (Adams 2007).

Statistical analysis
The experiments were arranged as completely randomized design with eight replications of each treatment and all the statistical analyses were performed by using OPSTAT statistical software package (Sheoran et al. 1998) at 0.05 probability level.

Essential oil content (%) in flowers with herb and without herb
Essential oil content (%) was found to be more in flowers without herb (0.15-0.18%) compared to flowers with herb (0.06-0.09%) during the day (Table  1). Data showed that in both cases the essential oil content increased as the days proceeded. There was a significant difference between harvesting time treatments in terms of essential oil content (%) ( Table 1). Essential oil content varied from 0.06%-0.09% and 0.15%-0.18% in fresh flowers with herb and without herb, respectively. Essential oil content was significantly influenced by harvesting time and significantly highest essential oil content (0.09 & 0.09% and 0.18 & 0.18%) was obtained when flowers with herb and without herb were harvested at 1 P.M. and 6 P.M compared to 6 A.M. (0.06 & 0.15%). In general, the results showed that the essential oil content in chamomile flowers both with herb and without herb increased from morning to evening. Higher essential oil content at 12 P.M. and 6 P.M. might be due to increased temperature and light intensity; higher light intensity and higher temperature might have enhanced metabolic reactions leading to the increased synthesis of secondary metabolites in the plant. Harvesting time is an important aspect for maximum oil recovery from the flowers (Oliveira et al. 2012). Salehi & Hazrati (2017) also reported similar results. Variation in oil yield at different harvesting time has been reported by various researchers in several plants (Marcum & Hanson 2006;Callan et al. 2007;Ebrahimi et al. 2008).

Influence of hydro-distillation duration on essential oil content in dried chamomile flowers
Distillation duration had significant effect on essential oil content which varied from 0.6%-1.2% (Table 2). The maximum essential oil content was recorded at 6 h distillation duration (1.2%) which was significantly higher as compared to hydrodistillation durations of 5 h (0.88%), 4 h (0.70%) and  (2001) also reported that distillation duration influences essential oil yield and its composition.

Essential oil composition as influenced by harvesting time
The qualitative and quantitative composition of the essential oil of chamomile flower was analysed by GC and GC-MS techniques. A total of 24 constituents were identified in chamomile essential oil samples extracted from flowers without herb and with herb at different harvesting times (Table 3). α-bisabolol oxide-A, α-bisabolone oxide-A, α-bisabolol oxide-B, (Z)-spiroether, chamazulene, and (E)-β-farnesene were marker compounds which were detected in the chamomile essential oil. Most representative fractions were dominated by oxygen containing sesquiterpenes which include most characteristic chamomile essential oil compounds such as α-bisabolol oxide-B, α-bisabolone oxide-A and α-bisabolol oxide-A. The results indicated that only flowers must be harvested for recovery of major compounds and herb part with flower should be avoided during harvesting. In comparison to chamomile oil in fresh flowers with herb, only chamomile oil of fresh flowers contains higher content of marker compounds. α-bisabolol oxide-B, (Z)-spiroether, and chamazulene were higher in the samples harvested at 12 P.M. (23.41%, 7.77%, 3.34%) and 6 P.M. (37.32%, 10.06%, 3.09%) and α-bisabolone oxide-A, α-bisabolol oxide-A were maximum in the samples harvested at 6 A.M.
(27.46%, 32.62%) and (E)-β-farnesene was found highest in the samples harvested at 12 P.M. (4.43%) ( Table 3). The variation in essential composition may be due to environmental factors such as light intensity, temperature and abiotic stresses (Tounekti & Khemira 2015). The results are in line with the findings of Salehi & Hazrati (2017). Chamazulene, α-bisabolol and its oxidised metabolites have powerful role in controlling the aroma of essential in chamomile which contribute to anti-inflammatory, anti-bacterial, insecticidal and anti-ulcer properties (Farhoudi et al. 2014). The presence and content of these compounds in chamomile essential oil indicate its drug value.

Essential oil composition as influenced by distillation duration
The essential oil obtained from German chamomile at different distillation duration (3, 4, 5 and 6 h.) was analyzed by GC-FID and GC-MS techniques. Altogether, 24 compounds represented 90.51 to 92.60% of the total essential oil (Table 4). α-bisabolol oxide-A (25.24% & 24.12%), α-bisabolol oxide-B (18.53% & 17.17%), (E)-β-farnesene (9.65% & 12.60%) and chamazulene (2.79% & 3.27%) were more in the essential oil samples which were hydro-distilled for 5 h and 6 h and (Z)-spiroether (10.32%) was more in those samples which were hydro-distilled only for 3 h. α-bisabolone oxide-A remained unchanged with increase in distillation duration. The maximum content of α-bisabolol oxide-A was obtained at 5 h distillation duration and further distillation decreased its content in the essential oil. Similar trend  was noticed in α-bisabolol oxide-B and α-bisabolone oxide-A. (E)-β-farnesene content increased as the distillation duration increased, it varied from 8.85% to 12.6%, maximum content was obtained at 6 h distillation duration. Chamazulene content was also maximum at 6 h (3.27%) and minimum at 3 h (2.27%) distillation duration. (Z)-spiroether showed decreasing trend with increased distillation duration, content decreased from 10.32% to 7.01% from 3 h to 6 h distillation duration, respectively. Verma et al. (2016) reported that distillation duration influenced the essential oil composition in Acorus calamus. Distillation duration influenced the essential oil yield and its composition in coriander spp. (Bruce et al. 2001).
The present study concluded that harvesting of (-)=Absent T 1 = hydro-distillation for 3h; T 2 =hydro-distillation for 4h; T 3 =hydro-distillation for 5h; T 4 =hydro-distillation for 6h Essential oil content and quality of German chamomile at varied harvesting and distillation time Salamon I 2007 Effect of the internal and external factors on yield and qualitative-quantitative characteristics of chamomile essential oil. In: Proceedings of the first international symposium on Chamomile research, flowers without herb part should be done during mid-day (12 P.M.) or in evening (6 P.M.) to get higher essential oil yield without affecting its quality and composition. In addition, hydro-distillation duration also showed positive effect on essential oil content. The maximum essential oil content was found in 6 h distillation followed by 5 h. Therefore, up to 5-6 h distillation of dried chamomile flowers is recommended for higher essential oil content. Distillation duration must be taken into consideration when comparing the essential oil yield and its composition.