www.nature.com/scientificreports OPEN Increasing medicinal and phytochemical compounds of conefower (Echinacea purpurea − + L.) as afected by NO 3 /NH4 ratio and perlite particle size in hydroponics Fatemeh Ahmadi1,3, Abbas Samadi1*, Ebrahim Sepehr1, Amir Rahimi2 & Sergey Shabala3 Medicinal plants are considered as one of the most important sources of chemical compounds, so preparing a suitable culture media for medicinal plant growth is a critical factor. The present study is aimed to improve the cafeic acid derivatives and alkylamides percentages of Echinacea purpurea − + root extract in hydroponic culture media with diferent perlite particle size and NO3 /NH4 ratios. Perlite particle size in the growing media was varied as very coarse perlite (more than 2 mm), coarse perlite (1.5–2 mm), medium perlite (1–1.5 mm), fne perlite (0.5–1 mm), and very fne perlite (less than 0.5 mm) in diferent ratios to peat moss (including pure perlite, 50:50 v/v, 30:70 v/v, and − + pure peat moss). Two NO3 /NH4 ratios (90:10 and 70:30) were tested in each growing media. All phytochemical analyses were performed according to standard methods using high performance liquid chromatography (HPLC). It was found that the E. purpurea grown in the medium containing very fne-grade perlite with 50:50 v/v perlite to peat moss ratio had the maximum cafeic acid derivatives, including chicoric acid (17 mg ­g−1 DW), caftaric acid (6.3 mg ­g−1 DW), chlorogenic acid (0.93 mg ­g−1 DW), cynarin (0.84 mg ­g−1 DW), and echinacoside (0.73 mg ­g−1 DW), as well as, alkylamides (54.21%). The percentages of these phytochemical compounds increased by decreasing perlite particle size and − + increasing of NO3 /NH4 ratio. The major alkylamide in the E. purpurea root extract was dodeca-2E, 4E, 8Z-10 (E/Z)-tetraenoic acid isobutylamide in all treatments, ranging from 31.12 to 54.21% of total dry weight. It can be concluded that optimizing hydroponic culture media and nutrient solution has signifcant efects on E. purpurea chemical compounds. Te Echinacea purpurea, widely known as purple conefower, is one of the popular medicinal plants of the Asteraceae family, from United States, Canada, Russia, and Australia. In recent years, it has gained international popularity due to claims that it benefcially stimulates the body’s immune system1,2. Extracts from the plant have shown antioxidative, antibacterial, antiviral, and antifungal properties, and are used in the treatment of the common cold, as well as respiratory and urinary diseases3,4. Cafeic acid derivatives, namely cafaric acid, chlo- rogenic acid, cynarin, echinacoside, and cichoric acid, are the main compounds of Echinacea spp. Of the cafeic acid derivatives, cichoric acid has been shown to possess immunostimulatory properties, promoting phagocyte activity both in vitro and in vivo. Besides, cichoric acid has exhibited antihyaluronidase activity, and a protective efect on the free radical-induced degradation of collagen. Cichoric acid has also been shown to have antiviral activity5,6 and, recently, inhibit HIV-1 integrase and replication 7,8. Echinacoside does not contribute towards the immunostimulant activity, but prospects collagen against reactive oxygen species, and also has antioxidant9, anti-infammatory, and cicatrizing activities10. Cafaric acid and chlorogenic acid play an important role in antiviral activity11, an efective free radical scavenging agent and preserver of collagen from free radical-induced 1Department of Soil Science, Faculty of Agriculture, Urmia University, Urmia, Iran. 2Department of Plant Production and Genetics, Faculty of Agriculture, Urmia University, Urmia, Iran. 3Tasmanian Institute of Agriculture, University of Tasmania, Hobart, TAS 7001, Australia. *email: [email protected] Scientifc Reports | (2021) 11:15202 | https://doi.org/10.1038/s41598-021-94589-4 1 Vol.:(0123456789) www.nature.com/scientificreports/ degradation10. Terefore, cafeic acid derivatives are widely measured as markers to determine the medicinal quality of E. purpurea extracts12. Te alkylamides, as a group of bioactive compound, has attached the most interest in terms of pharmacological activity13. Plants containing alkylamides are used as spices for their pungent and tingling sensations and are incorporated into topical cosmetics for their wrinkle-smoothing and anti-aging properties14. Tere is increasing evidence that lipophilic Echinacea preparations containing N-alkylamides can suppress stress-related cellular immune responses15. It was also demonstrated recently in several studies that alkylamides -containing Echinacea preparations trigger efects on the pro- infammatory cytokines16. Tus, it seems necessary to pay attention to efective strategies to improve the quality of E. purpurea phytochemical compounds. Diferent cultivation strategies have been developed for the production of E. purpurea. However, there are many considerations for plant production in greenhouse conditions, especially in hydroponic (or soilless) culture systems in recent years 17. Growing in hydroponic media may ofer several advantages over feld cultivation by controlling the plant nutrition requirement and facilitating the growth conditions18,19. Meanwhile, using artifcial substrates in the hydroponic cultivation system reduce the cost of establishing advanced cultivation systems, and it enables the farmer to make practical use of it by using commonly raw materials such as cocopeat, sand, and vermiculite as an initial plant growing media5,20. Nevertheless, diferent inorganic and organic products such as peat moss, perlite, mixed materials, etc., are fully or partially used instead of initial substrates due to their useful physical properties 20. Te particle size of substrates is a critical factor in air and water-holding capacity, root distribution, and plant growth, which are diferent based on their origin and preparation conditions7,21. A high volume of roots can concentrate at the top portion of the container includes low aeration and high water- holding capacity20. Attention to the chemical quality of hydroponic nutrient solution and its efect on yield and active compounds 22 + is so important . Of these nutrients, two major inorganic forms of nitrogen (N), the ammonium (NH 4 ) and − 23 the nitrate (NO3 ), can diferentially impact plant growth in many plant species . 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 NO 3 since NH 4 is toxic for plants and a few species grow well if NH 4 is the only source of N. − + It has been reported that using a mixture of NO3 and NH4 is optimum for nitrogen nutrient in most of the 23 − + crops . Previous research demonstrated that the NO3 /NH4 ratio could afect the morphological properties 18,24 23 24 − of E. purpurea . Zheng et al. and Ahmadi et al. reported that increasing NO 3 concentration in nutrient solution increased total phenolic and favonoid contents of E. purpurea root extract. However, the plant species − + 25 and environmental conditions are two critical factors that afect the optimum NO3 /NH4 ratio . So, the present research is focused on the development a hydroponic culture media with various perlite particle sizes and dif- − + ferent NO3 /NH4 ratios for improving the cafeic acid derivatives and alkylamides compounds of E. purpurea root extract at greenhouse conditions. Material and methods Growing conditions. Te experiment was accomplished in the research glass greenhouse at Urmia Uni- versity, Iran. Te plant growth conditions were controlled regularly inside the glass greenhouse (tempera- ture, humidity, and photosynthetic photon fux density (PPFD) were 22/18 (day/night) ± 1 °C, 70 ± 2%, and 650 ± 2 μmol m−2 s−1 respectively, and the length of the lighting period was 10 h photoperiod). All greenhouse conditions were controlled during the plant growth. Te E. purpurea seeds were purchased from an Iranian pri- vate joint-stock company, Pakan Bazr Esfahan (www. Pakanbazr.com; plant identifcation code: OTF-3). Tree seeds number were sowed in plastic cups flled with a mixture of perlite and peat mass 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 diferent ratios of perlite and peat mass as artifcial substrates (100% perlite (> 2 mm or > 10 U.S. mesh), 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 (< 35 U.S. mesh), 0.5–1 mm (35–18 U.S. mesh), 1–1.5 mm (18–14 U.S. mesh), 1.5–2 mm (14–10 U.S. mesh), and more than 2 mm (> 10 U.S. mesh). Te chemical composition of the nutrient solution based on Zheng et al.23 is shown in Table 1. Te pH and electrical conductivity (EC) of the nutrient solution were maintained between 5.7–6.2 and 1.0–1.5 dS m −1, respectively. According to the plant growth stage, 0.5–3.5 L day −1 was used in irriga- tion system23. Te E.purpurea grown at diferent culture media at the fowering stage were shown in Fig. 1. Sample preparation. Plants were harvested at the end of the fowering stage (8 months). Te plants were divided into roots, stems, fower heads, and lower and upper leaves afer washing with tap water. Root, sam- ples were dried at 25 ± ­1 °C, ground into a fne powder, and collected for phytochemical analysis26. Due to the perennial nature of this plant and the fowering of the plant in the second year, economically, fowers are frst considered as a medicinal organ and then the root. Of course, the root is the most important part and organ of medicine that can be grown in bioreactors. Total phenolic content. Measuring the total phenolic compounds in root samples was performed by the Folin–Ciocalteau method adapted from Singleton et al.27. In details, 10 μL of methanolic extracts and 1600 μL of distilled water was mixed, then 200 μL of Folin–Ciocalteau reagent (10% V/V prepared in distilled water) were added and lef at 25 °C for 5 min, then 200 μL of sodium carbonate (7.5%) was added and lef in a dark place at 25 °C for 30 min.