Magnetically treated water on phytochemical compounds of Rosmarinus officinalis L.

Irrigation using water treated with static magnetic field (SMF) has recently been used as a strategy to stimulate the growth and development of different plant species. The aim of this study was to characterize the bioactive compounds and evaluate the anatomical structure of Rosmarinus officinalis L. irrigated with SMF-treated water. Results demonstrate that the treatment promoted plant growth, the number of trichomes and increased concentrations of secondary metabolites. Methanol-extracted leaves revealed that rosmarinic acid was detected in both experimental groups, without a difference in the level. Camphor, α-terpineol and verbenone were determined as the most abundant compounds present in these leaf extracts and were strongly increased in plants irrigated with SMF-treated water. Similar results were also observed for endo-borneol, bornyl acetate and β-amyrin concentrations.Taken together, these results indicate that irrigation with SMF-treated water can be used to improve the production of rosemary to obtain pharmaceutical products with an increased antioxidative activity.

INTRODUCTION Rosemary (Rosmarinus officinalis L.) is an aromatic plant of the Lamiaceae family that is frequently used in medicine. It is an important source of polyphenols and is known for its high antioxidative activity (Suong et al., 2011).The most abundant bioactive compounds in rosemary leaves are phenols, monoterpenes, diterpenes and their derivatives, including carnosic acid and related stable compounds such as carnosol, rosmanol, epirosmanol and 7methylepirosmanol(Al-Sereiti et al., 1999). Rosemary isoften used for the production of natural antioxidant extracts andis reported to have a strong therapeutic potential in the treatment and prevention of many diseases including asthma,spasmogenicdisorders, liver disordersand hepatotoxicity, peptic ulcers, inflammatory diseases, ischemic heart disease, arteriosclerosis, Alzheimer and poor sperm motility (Suong et al., 2011;Fernández L.F. et al., 2014). Rosemary naturally grows throughout Cuba and is frequently found in home gardens. Recently, it has been removed from the Cuban National Formulations of Phytopharmaceuticals due toproblems associated with its cultivation. Indeed, rosemary has a poor vegetative propagation and low seed production. However, it is included inthe priority list of plants for the development of Natural and Traditional Medicine in Cuba(Report to the National Commission for the Development of Traditional and Natural Medicine, 2008), emphasizing the need to develop new rosemary cultivation strategies. Irrigation with magnetically treated water is an interesting strategy to improve rosemary production, as magnetic fields are known to strongly affect shoot growth and seed germination. Indeed, many reports have described the use of magnetically treated water and magnetic fields in agriculture. Its use has been associated with an increased plant metabolism (photosynthesis and water uptake) and improved plant growth and production. Irrigation using water treated with a static magnetic field (SMF)has been used for the cultivation of several plant species including tomato (Solanum lycopersicum L.), cucumber (Cucumis sativum L.), rice (Oriza sativum L.), faba bean (Vicia faba L.), snow pea (Pisum sativum L var. macrocarpon) and chickpea (Cicer arietinum L.) (Gesterberger P. et al., 1978;Gilart F. et al., 2013;Grewal H.S. et al., 2011a). This study aimed to characterize the bioactive compounds present in R. officinalis L. irrigated with SMF-treated water(100-150 mT)and to evaluate resulting changes in leaf histology underfield conditions.

Plant material
Rosmarinus officinalis L. plants were cultivated onan experimental plot in Santiago (Cuba) and the leaves were used to preparemethanol extracts. Voucher specimen isdeposited at the Herbarium of Biodiversity and Ecology Center (BIOECO) under accession number RB 21324. The experiment used an external magnetizer with permanent magnets designed, built and calibrated at the National Center of Applied Electromagnetism (NCAE).Magnetic induction ranged between 100 and 150 mT (Gilart F. et al., 2013). Plants were either irrigated with SMF-treated water(hereafter referred to as SMF plants) orwater not treated with SMF (referred to as control plants). Sixty plants were included in each treatment andgrown underthese conditionsfor 180 days. Irrigation was performed twice a day for 30 minutes through an air microjet system, consistingof aKSB ITUR pump and a valve-controlled system distributor. Irrigation was carried out using jets, which were set at aflow rate between 2.54 and 2.91 m 3 h -1 . Thewater velocityrangedbetween 1.4 and 1.6 ms -1 .

Scanning Electron Microscopy
The analysis of trichome morphology and numberwas performed in leaf samples fixed in formalin-acetic acidalcohol (FAA) solution (70%) (Johansen, 1940). For scanning electron microscopy (SEM), leaves were dehydrated in a graded ethanol series, submitted to critical point drying with CO2 (Leica EM CPD-030) and coated with a thin layer of gold (Denton Vacuum Desk IV, LLC). The samples were analyzed using a JEOL-JSM 6390 LV scanning electron microscope (Jeol USA Inc) as described byGesterberger P. et al. (1978).

Preparation of rosemary extracts
In order to prepare methanol leaf extracts, R. officinalis leaves were dried in an oven at 40°C for 72 h. Subsequently, 3 g of dried leaf sample was macerated in100 mL of methanol for 4 to 6 h in a Soxhlet device. The extract volume was then reduced to 10 mL in a Büchi rotoevaporator and centrifuged for 3 min at 3000 x g.The supernatant was filtered through a Whatman paper (GF/A, 110 mm)and stored at 4°C until further analysis.

Gas Chromatography-Mass Spectrometry (GC-MS)
Methanol extracts were analysed usinga GC-MS system (Agilent7890A/5975C GC-MS System) equipped with a JWMS5capillary column (Agilent Technologies;30mx 25mm x 0.25μm). The chromatographic conditions used is shown in Table 1. To visualize the components, plates were first sprayed with vanillin in ethanol (1%) and then with a solution of sulfuric acid in ethanol (10%). The plate was heated to110°C for 5 min and then analyzedunder white light for the evaluation of terpenoids and phenylpropanoids. Alternatively, plates were analyzedusing natural product reagent. In this case, they were first sprayed with 1% methanolic diphenyl boric acidb-ethylamine ester, followed by 5% ethanolic polyethylene glycol-4000 and then evaluated under UV light(365nm) for the detection of rosmarinic acid.For each of the bands observed, the retention factor (Rf) was calculatedas the ratio between the migration distance of the band and the migration distance of the solvent.

Trichomes in SMF plants
Glandular structures are known as primary sites of secondary metabolite biosynthesis, secretion and storage, and generally consist of either simple subcutaneous glands or trichomes.Plants of the Lamiaceaefamily presentboth capitate and peltate glandular trichomes. Both have the same basicmorphology, consisting of a basal region, a stalk anda head. Whereas capitate glandular trichomesare formed bya head with one secretory cell and a stalk containingtwo cells, the peltate type consists of a headwith eight secretory cells, one basal epidermal cell,and a wide unicellular stalk (Boix et al., 2011;Fahn, 1979). In R. officinalis, nonglandular trichomes arepresent on the veins and leaf margins andare diverse in morphology,anatomy and microstructure. Basically, they are classified according to their morphology. They canbe either unicellular ormulticellular, and unbranched or branched (Marin et al., 2006;Werker et al., 1985).
Results of this study show that glandular as well as nonglandular trichomes were observed in plants subjected to both irrigation treatments, although non-glandular trichomes were more numerous (Figure 1). The number of both trichome types on the abaxial leaf surface was higher in SMF plants ( Figure 1C-D) as compared to control plants ( Figure 1A-B). Leaves of control plants contained approximately six peltate trichomes per mm², whereas those of SMF plants contained 16 per mm² on average. The trichomes are primary sites for biosynthesis, secretion and storage of secondary metabolites (Taiz et al., 2015). Greater efficiency could then be expected in the production of bioactive compounds for the treated plants, with respect to the control in the relation structure. However, irrigation with SMF-treated water did not affect trichome structure, which is characterized by a prominent expandable cuticular layer (Boix et al., 2011).

Metabolites in SMF plants
The metabolites identifiedin methanol extractsof R. officinalis control plantsand SMF plants are listed in Table  2. Results show that irrigation with SMF-treated water strongly increased the levels of terpenoids including the bicyclic monoterpenes camphor, endo-borneol and bornyl acetate and the triterpene β-amyrin. Furthermore, terpenoids were the major class of bioactive compounds present in SMF plants (Table 2). Monoterpenes are produced in glandular trichomes, found on both leaf sides of R. officinalis (Boix et al., 2011).Therefore, the increased monoterpene levels in leaves of SMF plants are possibly related to their higher number of glandular trichomes ( Figure 1).   (Rašković et al., 2014) verified the high free radical scavenging activity and hepatoprotective effects of essential oils of aerial plant parts. Interestingly, one of the most abundant compounds present in these oils was camphor. Furthermore, results of this study showed that bornyl acetate levels were strongly increased in SMF plants as compared to control plants (Table 2). Recent studies showed that bornyl acetate lowered the production of lysophosphatidylcholine (LPS)-induced proinflammatory cytokines suchas TNF--1b, and IL-6. Therefore, irrigation of rosemary with SMF-treated water could possibly increase its anti-inflammatory potential for the treatment of inflammatory processes such as rheumatoid arthritis and osteoarthritis (Yang et al., 2014). In addition, a decreasedlevel of vitamin E was found in SMF plants as compared to control plants ( Table 2). Vitamin E is a fat-soluble compound that is mainly localized in membranes, protecting phospholipids against oxidative degeneration by reactive oxygen species (ROS).Itis involved in plantprotection against oxidative damage under differentstress conditions including drought, atmospheric pollutants, photosensitizing fungal toxinsand chilling (Fryer, 1992).The lower concentration of this vitamin in SMF plantspossibly indicates that irrigation with SMF-treated water diminishesoxidative stress. Using TLC/HPTLC, the presence of rosmarinic acid was confirmedin methanol extracts of control plants andSMF plants (Figure2), The presence of this compound in R. officinalisextracts has already been reported by (Wagner H. et al., 1996). Rosmarinic acid is an ester of caffeic acid and has antioxidant, anti-inflammatory, antibacterial, antiangiogenic, antimutagenic and antiallergenic activities (Nunes et al., 2015).In methanolicextract obtained of control and with SMFplants therosmarinic acid level it was no difference between both conditions.Whereas rosmarinic acid was detected under both irrigation regimes, no differences were observed in the levels of this compound in plants of either group.  We hypothesize that the increased levels of secondary metabolites in SMF plants could be related to an effect of SMF-treated water on cell membrane characteristics, resulting in an altered cell metabolism. Indeed, (Formicheva et al. (1992))reported that SMF-treated water significantly induced cell metabolism and mitosis in meristematic cells of pea, lentil and flax. Furthermore, gene transcriptionplaying an important role in regulating cellular processesalso seems to be affected by irrigation with SMF-treated water in Pisum sativum and Cicer arietinum (Grewal and Maheshwari, 2011). The effects of irrigation with magnetically treated water on plant secondary metabolite levels could also be a consequence of an influence on hormone levels, as increases in gibberellin (GA3) and kinetin levels were observed in broad bean plants irrigated with SMF-treated water . The effects of irrigation with SMF-treated water on these processes could be caused by changes occurring in the physical and chemical properties of water after application of a static magnetic field (Grewal et al., 2011b).

IV. CONCLUSION
Overall, results of this study indicate that irrigation with SMF-treated water could be used as a strategy to increase secondary metabolite levels in Rosmarinus officinalis, thereby promoting its therapeutic potential.

ACKNOWLEDGMENT
We thank Dr. Fidel Gilart Gonzalez and MSc. Douglas De As Yero of CNEA for magnetic field characterization and Dr. J. D. Sepúlveda of the Universidad Autónoma Metropolitana (UAM) -Iztapalapa (Mexico) for scanning electronic microscopy. We also thank theVLIR/UO (Belgium/Cuba) international program and the Centre for Environmental Sciences in Diepenbeek for financial support.We are also grateful to the International Scholarship Program sponsored by the Mexican Secretary of State of Exterior Relations and University of Oriente (Mexico/Cuba) and specifically CINVESTAV/Irapuato for financial support.