Abstract
Medicinal plants represent a valuable commodity due to beneficial effects of their natural products on human health, prompting a need for finding 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 fine (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 nitrate (NO3−) to ammonium (NH4+) ratios (90:10 and 70:30) were tested for each growing media. The medium containing very fine-grade perlite and 50:50 v/v perlite to peat moss ratio was found to be most optimal and beneficial 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 NO3−/NH4+ ratio caused a significant 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 NO3−/NH4+ ratio. It can be concluded that decreasing the perlite particle size and increasing the NO3−/NH4+ ratio increased the plant growth parameters and essential oil compositions in E. purpurea.
Subject terms: Plant sciences, Medical research
Introduction
Medicinal plants and their beneficial effects on human health are well known in various cultures for centuries1. Echinacea is a medicinal plant that belongs to the family of Asteracea/Compositae and is native to much of the United States2,3. 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 flowering head4. All parts of the E. purpurea species, especially root and coneflower, are rich in useful medicinal compounds, prompting significant attention of researchers to this species5,6.
Using of E. purpurea essential oil in medicinal, cosmetic, and food industries is common in all over the world7. The effect of E. purpurea essential oils on antimicrobial properties has been proven in previous studies8. 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, δ-cadinene, germacrene D, and β-caryophllyene, as antifungal, antiviral and antibacterial agents9,10. Extracts of essential oil obtained from E. purpurea are efficient in pest control and could regulate insect population at different life stages11. Numerous studies have been focused on prominent insecticidal influence of E. purpurea essential oil compositions and found the better influence of them in comparing with chemicals or a potential source of insecticides11. The antibacterial activity of E.purpurea essential oil is also reported against different food pathogens and bacteria in food industry12,13.
While the industrial application of E. purpurea essential oils is well established, several factors such as weather changes, plant growth stage14, and method of cultivation may influence both the composition and production of E. purpurea essential oil15. The open field cultivation of E. purpurea has some significant limitations such as crop inconsistency, seed dormancy16, water stress regims17, microbes, heavy metal ions and other pollutants2 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 systems18. Growing in a greenhouse also offer an additional advantages of more effective control of plant nutrition19,20.
Different hydroponic cultivation methods, such as artificial substrate media, water culture, and nutrient film techniques have been reported for E. purpurea cultivation18,21. However, using artificial 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 media22. 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 capacity22.
In addition to the importance of substrates properties in the hydroponic culture system, attention to the chemical composition of nutrient solution is important22. In terms of chemical composition of nutrient solutions, two major inorganic forms of nitrogen (N), the NH4+ and the NO3−, can differentially impact the various plant properties, based on the plant species18. Although the assimilation and metabolism of NH4+ form require less energy than that of NO3− in plants, the majority of plant species grow better on NO3− since NH4+ is toxic for plants and a few species grow well if NH4+ is the only source of N4. The plant species and environmental conditions are two critical factors that affect the optimum NO3−/NH4+ ratio23. It has been reported that different N application rates could affect the essential oil compositions of peppermints (Mentha piperita L.)24. Previous researches also demonstrated that the inorganic N application rate and sources could affect the essential oil content of sweet basil (Ocimum basilicum L.) and forage maize (Zea mays L.)25.
Although many researches have been performed on hydroponic culture of E. purpurea, but the use of culture media with different perlite particle sizes, different NO3−/NH4+ ratios, and their effects on essential oil compositions of E. purpurea has been assessed for the first time in this study. So the main goal of this study was to investigate the growth parameters and essential oil compositions of E. purpurea growing in new hydroponic culture media with various perlite particle sizes and different NO3−/NH4+ ratios.
Materials and methods
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 flux density (PPFD) fluctuated from 550 to 750 μmol m−2 s−1 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 filled 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 artificial 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−1, respectively. According to the stage of the plant growth, 0.5 to 3.5 L day−1 was used in fertigation system18.
Table 1.
Chemical properties of nutrient solution.
| Element | Fertilizer type | Amount |
|---|---|---|
| Nitrogen (N) | (NH4)2SO4-KNO3-Ca(NO3)2 | 15 mM |
| Phosphorus (P) | H3PO3 | 1 mM |
| Potassium (K) | KNO3 | 6 mM |
| Calcium (Ca) | Ca(NO3)2 | 4 mM |
| Magnesium (Mg) | MgSO4·7H2O | 2 mM |
| Sulfur (S) | Sulfate fertilizers | 2 mM |
| Iron (Fe) | Fe-EDTA | 50 µM |
| Manganese (Mn) | Mn SO4·H2O | 9 µM |
| Copper (Cu) | CuSO4·5H2O | 0.3 µM |
| Zinc (Zn) | ZnSO4·7H2O | 0.8 µM |
| Boron (B) | H3BO3 | 15 µM |
| Molybdenum (Mo) | H24Mo7N6O24·4H2O | 0.11 µM |
Sample preparation
Plants were harvested at the end of the flowering stage (eight months). The plants were divided into roots, stems, flower heads, and lower and upper leaves after washing with tap water. Root, flower heads, and leaves samples were dried at 25 ± 1 °C, ground into a fine powder and collected for further analyses6.
Plant growth parameters
The main growth parameters such as plant height (cm), fresh root weight (g plant−1), total fresh leave weight (g plant−1), total biomass (g plant−1), and leaf area (cm2) were determined for each plant at the matured stage. The leaf area was measured by using leaf area meter. Chlorophylls a and b were determined using 0.5 g of dry sample, which was homogenized with 10 mL acetone. Homogenized samples were centrifuged at 10,000×g for 15 min at 4 °C2. 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 that26:
| 1 |
where C is the total chlorophyll contents in mg/L of acetone extract, A645, and A663 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 120 g powder samples (root, leaves, and flower head) at a 1:10 (g mL−1) ratio. The essential oil was extract based on the distillation procedure using a commercial Clevenger apparatus27.
Analysis of essential oil
The essential oil analysis was performed using gas chromatography (GC) with 30 m × 0.25 mm capillary column coated with 0.25 µm film; carrier gas, helium (He) with a flow rate of 32 cm s−1; injector temperature of 260 °C and injection volume 0.2 µL. The programming was carried out from 90 °C for 2 min rising at 7 °C min−1 to 180 °C, at 15 °C min−1 to 220 °C. Identifications of different components were made by library search program on monoterpenoids and sesquiterpenoids mass spectral database and by comparing retention time with those of reference samples27.
Gas chromatography–mass spectrometry
Gas Chromatography–Mass Spectrometry (GC–MS) spectra were recorded on a Varian-3400 model fitted with a fused silica capillary column (30 m × 0.25 mm i.d.) coated with 0.25 µm film. The GC was run from 60 to 250 °C at a programmed rate of 8 °C min−1, 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.
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), fine perlite (0.5–1 mm), and very fine perlite (less than 0.5 mm), two NO3−/NH4+ rations (90:10 and 70:30), and a mixture of peat moss with different size of perlite at 50:50 v/v and 30:70 v/v peat moss to perlite ratios and pure peat moss (100% by volume). Data were analyzed using Duncan's multiple range tests at P ≤ 0.01, using statistical analysis software (SAS, 9.4; SAS Institute, 2011) statistical program.
License for the collection of plant specimens
The authors declare that the collection of plant and seed specimens were according to authorized rules.
Complying with relevant institutional, national, and international guidelines and legislation
The authors declare that all relevant institutional, national, and international guidelines and legislation were respected.
Results
Plant growth parameters
Plant growth parameters of E. purpurea under different culture media and NO3−/NH4+ ratios at the full flowering stage are shown in Table 2 and Figs. 1 and 2.
Table 2.
Some morphological properties of E. purpurea growing on various culture media and NO3−/NH4+ ratio at the flowering stage.
| Culture media | NO3−/NH4+ ratio | Height | Total fresh leave weight | Fresh root weight | Total biomass | Total Chlorophyll | Leaf area |
|---|---|---|---|---|---|---|---|
| (cm) | (g plant−1) | (g plant−1) | (g plant−1) | (mg g−1 FW) | (cm2) | ||
| 100% Pe (> 2 mm) | 90:10 | 5.3n ± 0.27 | 1.3 ± 0.13 | 3.1s ± 0.16 | 4.4x ± 0.25 | 5.12 ± 0.11 | 5 ± 0.41 |
| 70:30 | 3.2n ± 0.21 | 1.5 ± 0.11 | 3.1s ± 0.11 | 4.1x ± 0.14 | 3.53 ± 0.032 | 4 ± 0.35 | |
| 100% Pt | 90:10 | 55h ± 2.9 | 10 ± 2.1 | 20p ± 3.9 | 30w ± 4.1 | 8.8 ± 0.15 | 20 ± 0.25 |
| 70:30 | 47jk ± 2.1 | 8.2 ± 1.1 | 16r ± 2.5 | 24y ± 3.2 | 6.6 ± 0.12 | 15 ± 0.14 | |
| 50% Pt + 50% Pe (< 0.5 mm) | 90:10 | 105a ± 6.1 | 40 ± 3.2 | 75a ± 4.6 | 116a ± 7.1 | 18.5 ± 0.11 | 60 ± 0.35 |
| 70:30 | 91d ± 4.2 | 28 ± 1.2 | 52d ± 3.2 | 80d ± 4.1 | 15.1 ± 0.11 | 51 ± 0.23 | |
| 50% Pt + 50% Pe (0.5–1 mm) | 90:10 | 98b ± 5.1 | 27 ± 2.1 | 53c ± 4.1 | 81c ± 6.1 | 16.2 ± 0.13 | 55 ± 0.15 |
| 70:30 | 71f. ± 3.2 | 21 ± 1.1 | 48f. ± 2.5 | 70i ± 3.6 | 13.2 ± 0.11 | 49 ± 0.11 | |
| 50% Pt + 50% Pe (1–1.5 mm) | 90:10 | 96bc ± 5.9 | 26 ± 2.1 | 50e ± 3.2 | 76e ± 5.2 | 14.6 ± 0.16 | 50 ± 0.15 |
| 70:30 | 82e ± 3.2 | 24 ± 1.2 | 43i ± 1.9 | 67jk ± 2.4 | 12.8 ± 0.11 | 42 ± 0.10 | |
| 50% Pt + 50% Pe (1.5–2 mm) | 90:10 | 91d ± 4.3 | 25 ± 1.1 | 45h ± 2.5 | 71hi ± 3.6 | 13.8 ± 0.14 | 43 ± 0.11 |
| 70:30 | 71f. ± 2.1 | 24 ± 1.1 | 40k ± 1.9 | 65lm ± 2.4 | 12.2 ± 0.12 | 38 ± 0.11 | |
| 50% Pt + 50% Pe (> 2 mm) | 90:10 | 85e ± 3.3 | 23 ± 2.1 | 41jk ± 2.5 | 64m ± 4.1 | 13.2 ± 0.12 | 40 ± 0.14 |
| 70:30 | 66g ± 2.5 | 21 ± 1.1 | 37l ± 2.1 | 58p ± 3.5 | 11.5 ± 0.11 | 32 ± 0.10 | |
| 30% Pt + 70% Pe < 0.5 mm) | 90:10 | 85.2e ± 2.8 | 23 ± 1.1 | 50e ± 3.1 | 74g ± 4.1 | 16.1 ± 0.13 | 42 ± 0.15 |
| 70:30 | 71.6f. ± 2.1 | 22 ± 1.1 | 43i ± 2.4 | 65l ± 3.6 | 12.3 ± 0.11 | 35 ± 0.11 | |
| 30% Pt + 70% Pe (0.5–1 mm) | 90:10 | 71.1f ± 3.5 | 21 ± 1.3 | 47g ± 2.2 | 68j ± 4.1 | 13.5 ± 0.12 | 38 ± 0.15 |
| 70:30 | 63.9g ± 1.9 | 20 ± 1.1 | 37l ± 2.1 | 58p ± 3.1 | 10.4 ± 0.11 | 30 ± 0.11 | |
| 30% Pt + 70% Pe (1–1.5 mm) | 90:10 | 66.1g ± 2.5 | 18.8 ± 2.1 | 42ij ± 2.8 | 61no ± 4.2 | 12.9 ± 0.13 | 32 ± 0.13 |
| 70:30 | 52.7hi ± 2.1 | 15.4 ± 1.3 | 32n ± 1.6 | 47s ± 2.6 | 9.4 ± 0.10 | 25 ± 0.11 | |
| 30% Pt + 70% Pe (1.5–2 mm) | 90:10 | 55.5h ± 3.2 | 18.6 ± 1.2 | 34m ± 2.5 | 53q ± 3.1 | 11.3 ± 0.14 | 28 ± 0.13 |
| 70:30 | 43.1kl ± 2.1 | 14.1 ± 1.1 | 29° ± 1.1 | 43tu ± 2.1 | 8.4 ± 0.11 | 23 ± 0.11 | |
| 30% Pt + 70% Pe (> 2 mm) | 90:10 | 49.3ij ± 4.2 | 15.9 ± 2.5 | 32n ± 2.1 | 48rs ± 3.6 | 10.4 ± 0.13 | 25 ± 0.14 |
| 70:30 | 35.9m ± 2.1 | 12.7 ± 1.3 | 28° ± 1.5 | 41v ± 2.2 | 8.3 ± 0.11 | 19 ± 0.12 |
Pt: peat moss and Pe: perlite.
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 significantly 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 significant.
Figure 1.
Echinacea purpurea grown in (A) 100% peat moss, (B) 30% peat moss + 70% perlite (< 0.5 mm), (C) 50% peat moss + 50% perlite (< 0.5 mm), (D) 100% perlite (> 2 mm) culture media, just at 90:10 NO3−/NH4+ ratio. (All photos were taken by F. Ahmadi).
Figure 2.
Root morphology of E. purpurea grown in (A) 100% peat moss, (B) 30% peat moss + 70% perlite (< 0.5 mm), (C) 50% peat moss + 50% perlite (< 0.5 mm) at 90:10 NO3−/NH4+ ratio. The root of E. purpurea grown in 100% perlite was very small. (All photos were taken by F. Ahmadi).
Overall, plants grown in the 50% perlite + 50% peat moss medium with perlite particle size less than 0.5 mm and 90:10 NO3−/NH4+ ratio had the highest height (mean 105 cm) (Fig. 1), fresh leave weight (mean 30 g plant−1), fresh root weight (mean 65 g plant−1) (Fig. 2), total biomass (mean 96 g plant−1), and leaf area (mean 60 cm2). Decreasing perlite percentage of culture media and perlite particle size improved all the morphological properties (Table 2). There were significant differences in the plant morphological properties at different NO3−/NH4+ ratios. Increasing NO3− proportion in the N nutrition of E. purpurea caused to increase in plant height and root weight considerably (Table 2).
Essential oil analysis
The flower 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 NO3−/NH4+ ratios (90:10 and 70:30) are shown in Tables 3 and 4, respectively.
Table 3.
Essential oil chemical composition of E.purpurea grown at the best growing media at 90:10 NO3-/NH4+ ratio.
| Components | Class a | LRI b | KI | Percentage | ||
|---|---|---|---|---|---|---|
| Flower heads | Leaves | Root | ||||
| Heptane | NT | 776 | 732.68 | 0.018 | 0.013 | tr c |
| Myrcene | MH | 921 | 773.24 | 15 | 11 | 1.1 |
| (Z)-3-Hexenol acetate | NT | 930 | 794.80 | 0.32 | 0.25 | 0.11 |
| n-Tridecene | NT | 940 | 811.30 | 0.028 | 0.012 | tr |
| δ-Elemene | SH | 965 | 832.24 | 0.12 | 0.097 | tr |
| Cyclosativene | SH | 968 | 837.15 | 0.32 | 0.25 | 0.11 |
| α-Ylangene | SH | 983 | 858.63 | 0.19 | 0.13 | 0.12 |
| α-Copaene | SH | 998 | 906.21 | 1.4 | 1.1 | 0.25 |
| α-Pinene | SH | 1003 | 919.32 | 12 | 8.1 | 1.15 |
| β-Bourbonene | SH | 1010 | 923.68 | 0.22 | 0.19 | 0.064 |
| β-Cubebene | SH | 1018 | 969.90 | 0.19 | 0.17 | 0.013 |
| β-Elemene | SH | 1020 | 993.55 | 0.33 | 0.24 | 0.072 |
| n-Tetradecene | NT | 1022 | 1022.15 | 0.96 | 0.52 | 0.15 |
| β-Caryophyllene | SH | 1031 | 1076.38 | 11 | 7.6 | 1.1 |
| β-Copaene | SH | 1047 | 1093.50 | 1.1 | 0.78 | 0.15 |
| γ-Elemene | SH | 1074 | 1110.86 | 0.41 | 0.35 | tr |
| trans-α-bergamotene | SH | 1362 | 1143.80 | 0.78 | 0.43 | 0.096 |
| Aromadendrene | SH | 1365 | 1159.81 | 0.41 | 0.38 | tr |
| α-Humulene | SHS | 1370 | 1205.62 | 0.21 | 0.15 | tr |
| cis-Muurola-4(14), 5- diene | SH | 1390 | 1232.56 | 0.063 | 0.022 | tr |
| (Z)-8-dodecen-1-ol | NT | 1391 | 1240.25 | 0.014 | 0.027 | tr |
| Germacrene D | SH | 1412 | 1320.39 | 51 | 43 | 1.6 |
| (E)-B-ionone | AC | 1422 | 1329.12 | 0.28 | 0.21 | tr |
| 1-Pentadecene | NT | 1430 | 1360.85 | 4.4 | 2.1 | 0.91 |
| Bicyclogermacrene | SH | 1441 | 1372.13 | 0.66 | 0.46 | tr |
| α-Muurolene | SH | 1468 | 1390.23 | 1.2 | 0.89 | 0.031 |
| n-Pentadecane | NT | 1476 | 1396.14 | 0.58 | 0.38 | tr |
| (Z)-a-Bisabolene | SH | 1479 | 1400.09 | 0.43 | 0.35 | tr |
| trans-β-Guaiene | SH | 1488 | 1423.29 | 0.11 | 0.082 | tr |
| (E, E)-α-Farnesene | SH | 1415 | 1439.43 | 0.062 | 0.024 | tr |
| α-Bulnesene | SH | 1520 | 1452.14 | 0.14 | 0.097 | tr |
| δ-Amorphene | SH | 1548 | 1490.33 | 0.19 | 0.85 | 0.053 |
| trans-γ-Cadinene | SH | 1561 | 1513.77 | 0.057 | 0.032 | tr |
| δ-Cadinene | SH | 1570 | 1521.36 | 0.092 | 0.015 | tr |
| Selina-3,7(11)-diene | SH | 1575 | 1525.44 | 0.083 | 0.021 | tr |
| Germacrene B | SH | 1620 | 1537.14 | 0.054 | 0.013 | tr |
| Germacrene D-4-ol | OS | 1630 | 1540.32 | 0.092 | 0.042 | tr |
| Nerolidol acetate | OS | 1662 | 1554.56 | 0.063 | 0.011 | tr |
aClass: AC apocarotenoids; MH monoterpene hydrocarbons; SH sesquiterpene hydrocarbons; NT non-terpenes; OS oxygenated sesquiterpenes.
bLRI Linear Retention Index.
cTrace (below 0.01%); KI Kovats Index.
Table 4.
Essential oil chemical composition of E.purpurea grown at the best growing media at 70:30 NO3-/NH4+ ratio.
| Components | Classa | LRIb | KI | Percentage | ||
|---|---|---|---|---|---|---|
| Flower heads | Leaves | Root | ||||
| Heptane | NT | 776 | 732.68 | tr | tr | tr c |
| Myrcene | MH | 921 | 773.24 | 10 | 8.9 | 0.75 |
| (Z)-3-Hexenol acetate | NT | 930 | 794.80 | 0.21 | 0.11 | 0.083 |
| n-Tridecene | NT | 940 | 811.30 | tr | tr | Tr |
| δ-Elemene | SH | 965 | 832.24 | 0.082 | 0.023 | Tr |
| Cyclosativene | SH | 968 | 837.15 | 0.21 | 0.15 | Tr |
| α-Ylangene | SH | 983 | 858.63 | 0.11 | 0.08 | 0.092 |
| α-Copaene | SH | 998 | 906.21 | 0.89 | 0.66 | 0.11 |
| α-Pinene | SH | 1003 | 919.32 | 8.5 | 6.1 | 0.75 |
| β-Bourbonene | SH | 1010 | 923.68 | 0.15 | 0.091 | Tr |
| β-Cubebene | SH | 1018 | 969.90 | 0.12 | 0.024 | Tr |
| β-Elemene | SH | 1020 | 993.55 | 0.18 | 0.11 | 0.022 |
| n-Tetradecene | NT | 1022 | 1022.15 | 0.59 | 0.39 | Tr |
| β-Caryophyllene | SH | 1031 | 1076.38 | 7.1 | 5.5 | 0.45 |
| β-Copaene | SH | 1047 | 1093.50 | 0.84 | 0.62 | 0.083 |
| γ-Elemene | SH | 1074 | 1110.86 | 0.22 | 0.11 | Tr |
| trans-α-bergamotene | SH | 1362 | 1143.80 | 0.54 | 0.42 | Tr |
| Aromadendrene | SH | 1365 | 1159.81 | 0.21 | 0.15 | Tr |
| α-Humulene | SHS | 1370 | 1205.62 | 0.13 | 0.053 | Tr |
| cis-Muurola-4(14), 5-diene | SH | 1390 | 1232.56 | tr | tr | Tr |
| (Z)-8-dodecen-1-ol | NT | 1391 | 1240.25 | tr | tr | Tr |
| Germacrene D | SH | 1412 | 1320.39 | 47 | 33 | 0.95 |
| (E)-B-ionone | AC | 1422 | 1329.12 | 0.15 | 0.042 | Tr |
| 1-Pentadecene | NT | 1430 | 1360.85 | 3.1 | 1.8 | 0.25 |
| Bicyclogermacrene | SH | 1441 | 1372.13 | 0.25 | 0.16 | Tr |
| α-Muurolene | SH | 1468 | 1390.23 | 0.87 | 0.64 | 0.034 |
| n-Pentadecane | NT | 1476 | 1396.14 | 0.18 | 0.091 | Tr |
| (Z)-a-Bisabolene | SH | 1479 | 1400.09 | 0.23 | 0.15 | Tr |
| trans-β-Guaiene | SH | 1488 | 1423.29 | 0.052 | 0.014 | Tr |
| (E, E)-α-Farnesene | SH | 1415 | 1439.43 | tr | tr | Tr |
| α-Bulnesene | SH | 1520 | 1452.14 | 0.064 | 0.013 | Tr |
| δ-Amorphene | SH | 1548 | 1490.33 | 0.092 | 0.032 | Tr |
| trans-γ-Cadinene | SH | 1561 | 1513.77 | tr | tr | Tr |
| δ-Cadinene | SH | 1570 | 1521.36 | 0.023 | 0.014 | Tr |
| Selina-3,7(11)-diene | SH | 1575 | 1525.44 | tr | tr | Tr |
| Germacrene B | SH | 1620 | 1537.14 | tr | tr | Tr |
| Germacrene D-4-ol | OS | 1630 | 1540.32 | 0.017 | tr | Tr |
| Nerolidol acetate | OS | 1662 | 1554.56 | 0.019 | tr | Tr |
aClass: AC apocarotenoids; MH monoterpene hydrocarbons; SH sesquiterpene hydrocarbons; NT non-terpenes; OS oxygenated sesquiterpenes.
bLRI Linear Retention Index.
cTrace (below 0.01%); KI Kovats Index.
The essential oils were separated into 51 components, 38 of them were identified, 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 NO3−/NH4+ ratios (Tables 3 and 4).
The most abundant terpenes including, germacrene D, myrcene, α-Pinene, β-caryophyllene, and 1-pentadecene were found in chemical composition of E. purpurea extract by previous researchers. Comparing of the results in present study with other researches shows the noticeable increase in essential oil composition by using novel growing media and nutrition pattern (Table 5), which is related to improve physical properties of growing media (50% perlite + 50% peat moss medium with perlite particle size less than 0.5 mm and 90:10 NO3−/NH4+ ratio).
Table 5.
Maximum percentage of major essential oil compositions of E.purpurea reported in various previous studies.
| Germacrene D | Myrcene | α-Pinene | β-Caryophyllene | 1-Pentadecene | |
|---|---|---|---|---|---|
| (%) | |||||
| Present study | 51 | 15 | 12 | 11 | 4.4 |
| Diraz et al. (2012) | 11 | – | – | 7.2 | – |
| Sitarek et al. (2017) | 19 | 0.12 | 1.1 | – | 0.73 |
| Thappa et al. (2003) | 33 | 10 | 6.6 | 9.3 | 2.5 |
| Nyalambisa et al. (2016) | 20 | – | 3.7 | 4.5 | – |
| Hudaib and Cavrini (2002) | 29 | 1.7 | 2.3 | 3.1 | – |
| Holla et al. (2005) | 4.8 | 2.1 | 5.1 | 3.6 | 2.5 |
| Kyslychenko et al. (2008) | 25 | 11 | 7.5 | 5.2 | 2.9 |
| Kan et al. (2008) | 32 | 9.4 | 4.2 | 4.3 | 3.2 |
Discussion
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-filled porosity (AFP) of 33% and water holding capacity (WHC) of 56% in comparison with other fine-perlite culture media (Table 6). 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 significant effect of culture media on photosynthesizing pigments (Table 2). It has been reported that the application of N fertilizers in the fine 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 yield18,28.
Table 6.
Physical properties of media used in greenhouse E. purpurea culture.
| Culture media | Water holding capacity | Air-filled porosity | Bulk density | Total porosity |
|---|---|---|---|---|
| (% vol) | (%) | (g cm−3) | (%) | |
| 100% Pe (> 2 mm) | 56 | 33 | 0.16 | 89 |
| 100% Pt | 75 | 10 | 0.21 | 85 |
| 50% Pt + 50% Pe (< 0.5 mm) | 68 | 24 | 0.18 | 92 |
| 50% Pt + 50% Pe (0.5–1 mm) | 65 | 26 | 0.18 | 91 |
| 50% Pt + 50% Pe (1–1.5 mm) | 64 | 28 | 0.18 | 92 |
| 50% Pt + 50% Pe (1.5–2 mm) | 62 | 31 | 0.17 | 93 |
| 50% Pt + 50% Pe (> 2 mm) | 60 | 33 | 0.17 | 93 |
| 30% Pt + 70% Pe (< 0.5 mm) | 61 | 28 | 0.14 | 89 |
| 30% Pt + 70% Pe (0.5–1 mm) | 58 | 30 | 0.14 | 88 |
| 30% Pt + 70% Pe (1–1.5 mm) | 56 | 32 | 0.15 | 88 |
| 30% Pt + 70% Pe (1.5–2 mm) | 53 | 33 | 0.15 | 86 |
| 30% Pt + 70% Pe (> 2 mm) | 50 | 35 | 0.16 | 85 |
The numbers in the parentheses show perlite particle size.
Pt peat moss and Pe perlite.
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 flower head than leave and root at both NO3−/NH4+ 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 flower head and 0.95% in root to 47% in flower head at 90:10 and 70:30 NO3−/NH4+ ratios, respectively. Variability was also obtained in the concentration of other compositions. The results (Tables 3 and 4) indicate that the various components of the essential oil of E. purpurea are specific to the plant organs, which influence their concentration.
The variations in the concentrations of various essential oil compositions at different NO3−/NH4+ ratios (Tables 3 and 4) may be due to supply different amounts of NO3− to the plant. The presence of N as a key factor can affect the production of essential oils in aromatic plants29. Nitrogen is critical factor in biosynthesis pathway of essential oil in medicinal and aromatic plants30. Nitrogen increases photosynthetic efficiency 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 metabolism31. 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 pathway32. Besides, essential oils are terpenoids compounds whose constituent units (isonoids) such as isopentenyl pyrophosphate and dimethyl ally pyrophosphate are strongly formed into adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH), and due to the effect of N in the production of these compounds, the amount of essential oil increased33. Nitrogen increases the essential oil content of plants by increasing the dry weight (Nyalambisa et al. 2016). Comparing of the results in Tables 3 and 4 indicated that increase of NO3− concentration could increase the percentage of essential oil composition due to its effect on essential oil biosynthesis as demonstrated in previous researches34.
Germacrene D, myrcene, α-Pinene, β-Caryophyllene, and 1-Pentadecene were the major compositions of essential oil of E.purpurea grown in very fine-grade (< 0.5 mm) perlite with 50:50 v/v perlite to peat moss ratio (Tables 3 and 4). The compositions have a valuable beneficial effects in medicine and agriculture industries7.
Germacrene D is a natural hydrocarbon, belongs to sesquiterpenes, which is found in aromatic plants27. The hydrocarbon is a useful bioactive phytochemical compound in human health Maintains healthy blood pressure is one of the important roles of germacrene compounds in humans8. The antimicrobial properties of germacrene D were reported in previous researches10. Anti-inflammatory, antimicrobial, and antioxidant effects of germacrene D are also well known8. The anti-insect influence of germacrene D has been reported in previous studies10. Myrcene is a terpene with anti-inflammatory and anti-depressant effects14. Regulating the efficiency of other terpenes and cannabinoids by increasing of myrcene is recognized previously7. Pinene has a several of potential benefits, including anti-inflammatory, antimicrobial, antitumor, antioxidant, and neuroprotective effects. It may also help counteract the short-term memory issues that many people experience. Beta-caryophyllene is also known for antioxidant and anti-inflammatory medicinal effects. It is especially useful to decrease pain and anxiety35.
It was found that the mixture of peat moss into very fine-grade perlite (< 0.5 mm) at 50:50 v/v perlite to peat moss ratio had a significant increase in plant growth parameters, which increased by increasing of NO3−/NH4+ ratio. The essential oil content was significantly highest in the 50:50 v/v perlite to peat moss ratio (perlite particle size less than 0.5 mm) than others. The major terpene hydrocarbons found in extract of E. purpurea with the best growth parameters were germacrene D, myrcene, α-pinene, β-caryophyllene, and 1-Pentadecene, respectively. The percentages of these terpene hydrocarbons were increased by increasing of NO3−/NH4+ ratio. Using of perlite and peat moss mixture for plant cultivation not only affects the plant growth parameters and essential oil compositions, but also reduces production costs in hydroponic systems.
Acknowledgements
The authors are thankful to Mr. Yahya Hasirchi for preparing the experimental facilities and the Office of Vice Chancellor for Research and Technology, Urmia University.
Author contributions
F.A. performed the experiment and wrote the paper, A.S. conceived the idea, E.S. and A.R. reviewed the collected data, and S.S. and A.S. edited the paper. A.S. was responsible for editing, original data and text preparation. All authors took responsibility for the integrity of the data that is present in this study.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Contributor Information
Abbas Samadi, Email: asamadi2@gmail.com.
Amir Rahimi, Email: e.rahimi@urmia.ac.ir.
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