Improving Essential Oil Compositions of Purple Cone�ower (Echinacea Purpurea L.) Medicinal Plant Using Novel Growing Media and Nutrition Pattern in Hydroponics

Medicinal plants represent a valuable commodity due to bene�cial effects of their natural products on human health, prompting a need for �nding a way to optimize/increase their production. In this study, a novel growing media with various perlite particle size and its mixture with peat moss was tested for hydroponic-based production of Echinacea purpurea medicinal plant under greenhouse conditions. The plant growth parameters such as plant height, total fresh leave weight, fresh root weight, total biomass, total chlorophyll, leaf area, and essential oil compositions were assessed. Perlite particle size in the growing media was varied from very coarse (more than 2 mm) to very �ne (less than 0.5 mm), and the ratio between perlite and peat moss varied from 50:50 v/v to 30:70 v/v. In addition, two NO 3-/NH 4+ ratios (90:10 and 70:30) were tested for each growing media. The medium containing very ne-grade perlite and 50:50 v/v perlite to peat moss ratio was found to be most optimal and bene�cial for E. purpurea performance, resulting in maximal plant height, fresh and dry weight, leaf surface area, and chlorophyll content. It was also found that an increase in NO 3 - /NH 4 + ratio caused a signi�cant increase in plant growth parameters and increase the plant essential oil content. The major terpene hydrocarbons found in extract of E. purpurea with the best growth parameters were germacrene D (51%), myrcene (15%), α-pinene (12%), β-caryophyllene (11%), and 1-Pentadecene (4.4%), respectively. The percentages of these terpene hydrocarbons were increased by increasing of NO 3-/NH 4+ ratio. It can be concluded that decreasing the perlite particle size and increasing the NO 3-/NH 4+ ratio increased the plant growth parameters and essential oil compositions in E. purpurea.


Introduction
Medicinal plants and their bene cial effects on human health are well known in various cultures for centuries (Waidyanatha et al., 2020).Echinacea is a medicinal plant that belongs to the family of Asteracea/Compositae and is native to much of the United States (Ahmadi et al., 2020).The most popular species of the plant in medicine are E. purpurea, E. angustifolia, and E. pallida.The species has a black and pungent root and purple coneshape owering head (Ahmadi et al., 2020).All parts of the E. purpurea species, especially root and cone ower, are rich in useful medicinal compounds, prompting signi cant attention of researchers to this species (Senica et al., 2019).
Using of E. purpurea essential oil in medicinal, cosmetic, and food industries is common in all over the world (Maggini et al., 2019).The effect of E. purpurea essential oils on antimicrobial properties has been proven in previous studies (Balciunaite et al., 2020).Also accepted is the role of some constituents of the essential oil of E. purpurea, including α-phellandrene, myrcene, limonene, α-pinene, β-pinene a, δcadinene, germacrene D, and β-caryophllyene, as antifungal, antiviral and antibacterial agents (Shari -Rad et al., 2018).Extracts of essential oil obtained from E. purpurea are e cient in pest control and could regulate insect population at different life stages (Clifford et al., 2002).Numerous studies have been focused on prominent insecticidal in uence of E. purpurea essential oil compositions and found the better in uence of them in comparing with chemicals or a potential source of insecticides (Clifford et al., 2002).The antibacterial activity of E.purpurea essential oil is also reported against different food pathogens and bacteria in food industry (Indras et al., 2020).
While the industrial application of E. purpurea essential oils is well established, several factors such as weather changes, plant growth stage (Mousavi et al., 2019), and method of cultivation may in uence both the composition and production of E. purpurea essential oil (Letchamo et al., 2002).Of speci c interest is a cultivation method.The open eld cultivation of E. purpurea has some signi cant limitations such as crop inconsistency, seed dormancy (Karg et al., 2019), water stress regims (Darvizheh et al., 2019), microbes, heavy metal ions and other pollutants (Ahmadi et al., 2020) and loss of wild germplasm, that affect the different chemical composition of the plant extract.The above limitations have prompted a shift towards plant production under greenhouse conditions, especially in hydroponic (or soilless) culture systems (Zheng et al., 2006).Growing in a greenhouse also offer an additional advantages of more effective control of plant nutrition (Russo et al., 2019).
Different hydroponic cultivation methods, such as arti cial substrate media, water culture, and nutrient lm techniques have been reported for E. purpurea cultivation (Zheng et al., 2006).However, using arti cial substrates in the hydroponic cultivation system reduces the cost of establishing advanced hydroponic cultivation systems and also enables the farmer to make a practical use of it by using commonly raw materials such as cocopeat, sand, and vermiculite as an initial plant growing media (Samadi, 2011).Nevertheless, different inorganic products such as peat moss, perlite, mixed materials, etc. are fully or partially used instead of initial substrates due to their useful physical properties.The particle size of substrates is a critical factor in air and water-holding capacity, root distribution, and plant growth, which are different based on their origin and preparation conditions.A high volume of roots can concentrate at the top portion of the container includes low aeration and high water-holding capacity (Samadi, 2011).
In addition to the importance of substrates properties in the hydroponic culture system, attention to the chemical composition of nutrient solution is required (Samadi, 2011).Of speci c interest is a source of nitrogen for plant cultivation.It has been reported that using a mixture of NO 3 -and NH 4 + can increase the leaf area, root distribution, total biomass, and plant growth parameters in comparison with each for nitrogen alone (Demirci et al., 2020).Previous researches also demonstrated that the NO

Growth conditions
The experiment was performed in a commercial greenhouse at Urmia University, West Azerbaijan, Iran.The air temperature was 22/18 ̊ C (day/night) and the humidity ranged from 70 to 80%.The maximum photosynthetic photon ux density (PPFD) uctuated from 550 to 750 μmol/ m 2 s inside the greenhouse.
The E. purpurea seeds were purchased from Iranian private joint-stock company, Pakan Bazr Esfahan (www.Pakanbazr.com).The seeds were sowed in plastic cups lled with a mixture of perlite and peat moss substrates as a medium to initiate germination.Irrigation was performed based on greenhouse conditions regularly.Seedlings (with four real leaves) were translocated to experimental plastic pots (2.5 L) containing a different ratios of perlite and peat moss as arti cial substrates (100% perlite, 100% peat moss, 50% (v) perlite + 50% (v) peat moss, 70% (v) perlite +30% (v) peat moss) with various perlite particle size containing less than 0.5 mm, 0.5-1 mm, 1-1.5 mm, 1.5-2 mm, and more than 2 mm.Chemical concentrations of nutrient solution are shown in Table 1.The pH and electrical conductivity (EC) of the nutrient solution were maintained between 5.7 to 6.2 and 1.0 to 1.5 dS/m, respectively.According to the stage of the plant growth, 0.5 to 3.5 L/day was used in fertigation system (Zheng et al., 2006).

Sample preparation
Plants were harvested at the end of the owering stage (eight months).The plants were divided into roots, stems, ower heads, and lower and upper leaves after washing with tap water.Root, ower heads, and leaves samples were dried at 25 ± 1 °C, ground into a ne powder and collected for phytochemical analysis (Senica et al., 2019).

Plant growth parameters
The main growth parameters such as plant height (cm), fresh root weight (g/plant), total fresh leave weight (g/plant), total biomass (g/plant), and leaf area (cm 2 ) were determined for each plant at the matured stage.The leaf area was measured by using leaf area meter (model AccuPAR LP-80).Chlorophylls a and b were determined using 0.5 g of dry sample, which was homogenized with 10 mL acetone.Homogenized samples were centrifuged (Gyrozen ® , South Korea) at 10000 x g for 15 min at 4 °C (Ahmadi et al., 2020).The supernatant was separated, and the absorbance spectra were measured at 400-700 nm.The total chlorophyll was calculated at 645 nm and 663 nm respectively.So that (Arnon, 1949): Where C is the total chlorophyll contents in mg/L of acetone extract, A 645 , and A 663 are the absorption of the extract at 645 and 663 nm.

Extraction of essential oils
The E. purpurea plants which shown the best morphological properties (maximum height, dry and wet weight of leaves and roots, and leaf area) were selected for analysis of essential oil.Distilled water was added to 20 g powder samples ( ower heads at the matured stage, leave, and root) at a 1:10 (g/mL) ratio.
The essential oil was extract based on the distillation procedure using a commercial Clevenger apparatus (Kaya et al., 2019).

Analysis of essential oil
The essential oil analysis was performed using gas chromatography (GC, Shimadzu 9A) with 30 m × 0.

6. Gas Chromatography-Mass Spectrometry
GC-MS spectra were recorded on a Varian-3400 model tted with a fused silica capillary column (30 m × 0.25 mm i.d.) coated with 0.25µm lm.The GC was run from 60 -250 °C at a programmed rate of 8 °C /min, hold at 100 °C for 2 min, using He as the carrier gas at a pressure of 1.6 kg/cm 2 and injector temperature of 250 °C.The GC column was coupled directly to the quadrupole mass spectrometer operated in the electron impact (EI) mode at 70 eV.Mass spectra were recorded at a scan speed of 9 at m/z 700-10.

7. Statistical Analysis
The statistics was based on the factorial with completely randomize design with three replications.The factors contained different sizes of perlite, including very coarse perlite (more than 2 mm), coarse perlite (1.5-2 mm), medium perlite (1-1.5 mm), ne perlite (0.5-1 mm), and very ne perlite (less than 0. peat moss medium with perlite particle size less than 0.5 mm and 90:10 NO 3 -/NH 4 + ratio had the highest height (mean 105 cm) ( Figure 1), fresh leave weight (mean 30 g/plant), fresh root weight (mean 65 g/plant) (Figure 2), total biomass (mean 96 g/plant), and leaf area (mean 60 cm 2 ) (Figure 3).Decreasing perlite percentage of culture media and perlite particle size improved all the morphological properties (Table 2).There were signi cant differences in the plant morphological properties at different NO Based on open hydroponic cultivation system in the present experiment, decreasing perlite particle size, increased the retention time of nutrient solution in the culture media.Increasing nutrient accessibility for plant roots by increasing retention time improves nutrient uptake and plant growth.However, the pure perlite culture system (100% perlite, < 0.5 mm) has a very low air-lled porosity (AFP) of 33% and water holding capacity (WHC) of 56% in comparison with other ne-perlite culture media (Table 3).Accordingly, the lowest growth parameters were obtained in pure perlite medium (Table 2), which can be attributed to the rapid withdrawal of nutrient solution from the culture medium and the inability of the medium to maintain the nutrient solution.Due to the high porosity of peat mass and nutrient solution retention capability, an increase of the plant morphological parameters is expected in the presence of peat moss in various cultural media (Table 2).The noticeable increase in chlorophyll content by reducing perlite particle size implies the signi cant effect of culture media on photosynthesizing pigments (Table 2).It has been reported that the application of nitrogen fertilizers in the ne perlite culture media increased N content of the plants, thereby increasing their chlorophyll content, subsequently, and their ability to absorb sunlight and produce photosynthates, which resulted in their higher leaf area, and growth and yield (Coelho et al., 2020).

Essential oil analysis
The ower head, leaves, and root essential oil compositions of E. purpurea grown at the 50% perlite + 50% peat moss medium with perlite particle size less than 0.5 mm growing medium at different NO 3 -/NH 4 + ratios (90:10 and 70:30) are shown in Tables 3 and 4, respectively.The essential oils were separated into 51 components, 38 of them were identi ed, comprising 92.8% of the total essential oil yield (Tables 3 and  4).
The content and composition of the essential oil exhibited a variable pattern at different plant organs at different NO 3 -/NH 4 + ratios (Tables 4 and 5).The essential oil was characterized by a higher percentage of terpene hydrocarbons, especially the monoterpenoids, which constituted 60 to 70% of the essential oil composition.The major terpene hydrocarbons found are α-pinene, myrcene, β-caryophyllene, 1-Pentadecene, and germacrene D. The percentages of these terpene hydrocarbons were higher in ower head than leave and root at both NO 3 -/NH 4 + ratios.The most abundant terpene found in the essential oil was germacrene D, which showed a remarkable rise from 1.5% in root to 51% in ower head and 0.95% in root to 47% in ower head at 90:10 and 70:30 NO 3 -/NH 4 + ratios, respectively.Variability was also obtained in the concentration of other compositions.The results (Tables 4 and 5) indicate that the various components of the essential oil of E. purpurea are speci c to the plant organs, which in uence their concentration.
The variations in the concentrations of various essential oil compositions at different NO 3 -/NH 4 + ratios (Tables 4 and 5) may be due to supply different amounts of N-NO 3 -to the plant.The presence of nitrogen as a key factor can affect the production of essential oils in aromatic plants (Oniszczuk et al., 2019).
Nitrogen is critical factor in biosynthesis pathway of essential oil in medicinal and aromatic plants ( Baričevič and Zupančič, 2002).Nitrogen increases photosynthetic e ciency and plays an important role in increasing the amount of essential oil by increasing the number and area of leave and providing a suitable condition for receiving sunlight energy and also participating in the structure of chlorophyll and enzymes involved in photosynthetic carbon metabolism (Hosseinpour et al., 2020).Nitrogen is an essential nutrient in plants used to synthesize many organic compounds in plants such as nucleic acids, enzymes, proteins, and amino acids, which are necessary for essential oil biosynthesis pathway (Banica et al., 2020).Besides, essential oils are terpenoids compounds whose constituent units (isonoids) such as isopentenyl pyrophosphate and dimethyl ally pyrophosphate are strongly formed into ATP and NADPH, and due to the effect of nitrogen in the production of these compounds, the amount of essential oil increased (Sitarek et al., 2017).Nitrogen increases the essential oil content of plants by increasing the dry weight (Nyalambisa et al., 2016).Comparing of the results in Tables 4 and 5 indicated that increase of NO 3 -concentration could increase the percentage of essential oil composition due to its effect on essential oil biosynthesis as demonstrated in previous researches (Diraz et al., 2012).
Germacrene D is a natural hydrocarbon, belongs to sesquiterpenes, which is found in aromatic plants (Kaya et  The numbers in the parentheses show perlite particle size Each value is expressed as mean ± SD (n= 3).Means bearing different letters in the same column are signi cantly different (P ≤ 0.01)

The numbers show as mean ± standard deviation
The interaction effect of different treatments on total fresh leave weight, chlorophylls a and b, and leaf area was not signi cant The numbers show as mean ± standard deviation The interaction effect of different treatments on total fresh leave weight, chlorophylls a and b, and leaf area was not signi cant Pt: peat moss and Pe: perlite The numbers in the parentheses show perlite particle size  Figure 2

Figures Figure 1
Figures (Kaya et al., 2019)mn coated with FFAP 0.25µm lm; carrier gas, helium (He) with a ow rate of 32 cm/s; injector temperature of 260 °C and injection volume 0.2µL.The programming was carried out from 90 °C for 2 min rising at 7 °C /min to 180 °C, at 15 °C/min to 220 °C.Identi cations of different components were made by library search program on monoterpenoids and sesquiterpenoids mass spectral database and by comparing RRT with those of reference samples(Kaya et al., 2019).

Table 2 .
Some morphological properties of E. purpurea growing on various culture media and NO 3 Pt: peat moss and Pe: perlite

Table 3 .
Physical properties of media used in greenhouse E. purpurea culture.

Table 6 .
Maximum percentage of major essential oil compositions of E.purpurea reported in various previous studies