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European Journal of Biotechnology and Bioscience European Journal of Biotechnology and Bioscience ISSN: 2321-9122 Impact Factor: RJIF 5.44 Received: 11-09-2018: Accepted: 13-10-2018 www.biosciencejournals.com Volume 6; Issue 5; September 2018; Page No. 52-55 Chrome elicitation for secondary metabolites stimulation in Hypericum adenotrichum Spach Ömer Yamaner1, Bengi Erdağ2* 1, 2 Department of Biology, Faculty of Arts & Sciences, Aydın Adnan Menderes Üniversity, 09010 Aydın, Turkey Abstract In this study, it was aimed to induction the hypericines (hypericin and pseudohipericin) and some flavonoids (hyperoside and isoquercitrin) by application of chrome as elicitor to the ın vitro seedlings of Hypericum adenotrichum Spach which an endemic and medicinal plant. Sterilised seeds were germinated in ¼ MS/Galzy medium. For elicitation, In vitro germinated seedlings were transferred to ¼ MS/Galzy medium containing 0.01 mM and 0.1 mM chrome two weeks after incubation. Seedlings were extracted with methanol after 15 and 30 days of application. The extacts were analysed by HPLC. The amounts of hyperoside and isoquercitrin increased by 1.7 and 1.8 fold at 0.01 mM chrome concentrations in 15 day treatment. There was no significant change in 0.01 mM chrome applications during 30 days of application. In both 15 and 30 day 0.1 mM chrome applications, flavonoid amounts decreased significantly. By the application of 0.01 mM of chrome for 15 days the amount of pseudohypericin incresed up to 2.2 fold. The amount of hypericin also increased up to 1.7 fold. 30 day chrome applications did not cause a significant change in the amount of hypericin. Keywords: Hypericum adenotrichum, chrome, elicitor, hypericins, flavonoids 1. Introduction [14]. Among the mentioned components, pseudohypericin and Plant secondary metabolites are defined as “a type of molecule hypericin have antidepressant, antimicrobial, antiviral and group that does not participate in primer biochemical patways antitumoral properties. It has been reported that exracts of in cell growth and proliferation, but plays a role, in particular, Hypericum adenotrichum have anticancer activity [15]. In in its environment adaptation” [1]. The accumulation of another study, Sarımahmut et. al. investigated genotoxic and secondary metabolites frequently occurs with different stress apoptotic potential of H. adenotrichum Spach [16]. Our types, with various elicitors and/or signal molecules [2] and is knowledge on the elicitation of secondary metabolites of H. regulated by evolution, genetics, growth conditions, adenotrichum is very limited and relies upon on a few studies. physiological variations, climate, photoperiod and light [3, 4]. In the first report, pectin and mannan stimulated hypericin and Initially, the term elicitor is used for molecules that can induce pseudohypericin production depending on concentration and the production of phytoalexins [5] but it is now widely used for treatment duration by using ın vitro developed seedlings [17]. compounds that stimulate all kind of plant defense [6]. Elicitors Polyethylene glycol and sucrose have the same result in the are essentially divided into three categories based on their second report [18]. origin, i.e. biological, chemical and physical triggers [7]. Some To date there is no report on the effect of chrome treatment on inorganic substances (e.g. heavy metals, metal oxides and stimulation of elicitation in secondary metabolites of metal ions) have potentials for acting as elicitors of plant Hypericum adenotrichum Spach. Present study investigates secondary metabolism [8, 9]. the effect of chrome application on secondary metabolite Plant secondary metabolism products have been used for contents of H. adenotrichum, an endemic species from Turkey centuries for therapeutic purposes. Among these, the to elucidate effectiveness of chrome as an elicitor. components of the Hypericum species have a special significance. Although studies on secondary metabolite 2. Materıal and methods content are present in different Hypericum species, studies are 2.1 Plant material usually focused on Hypericum perforatum L. [10-12]. The The seeds of H. adenotrichum plant were collected from manipulation of secondary metabolism of H. perforatum by Karıncalı Mountain (1400m. Karacasu, AYDIN; GPS: N 37º means of elicitation has attracted by a number of studies in 24´ 57,9 ´´, E 28º 20 ´ 25 ´´) where the plant was naturally recent years and these efforts provided an extended data pool distributed. The voucher specimens were deposited in the on the topic [7]. herbarium of Adnan Menderes Üniversity (AYDN-2396) Hypericum adenotrichum Spach is an endemic species in Turkey [13]. The plant has a capacity pharmaceutical 2.2 In vitro culture and elicitation importance due to the content of hyperforin, pseudohypericin, Seed sterilization and was carried out by the method hypericin, chlorogenic acid, routine, hyperoside, apigenin-7-0- recommended by Yamaner et al. [17]. The sterilized seeds were glucoside, quercitrin, quercetin, camphorol, amentoflavone cultured in glass jars containing micro and 1/4 MS macro 52 European Journal of Biotechnology and Bioscience mineral salts [19] Galzy vitamins [20], sucrose (30g/L). The pH between 0.05-10 μg/mL), hyperoside (five set of standard of media were adjusted to 5.8 before adding agar (agar-agar, dilutions, between 0.05-7.5 μg/mL), isoquercitrin (six set of 8g/L). The cultures were maintained in a growth chamber at standard dilutions, between 0.2-10 μg/mL). Linear regression 18 ± 1◦ C under 16-h light period. For elicitation, seedlings equations (R2 > 0.99) were obtained. Quantification of were transferred to same medium supplemented with 0.01 mM pseudohypericin, hypericin, hyperoside, and isoquercitrin was and 0.1 mM chrome two weeks after incubation. A control based on peak area (RT, retention time of 22.4, 25.0, 13.5, and treatment without chrome was also performed. The seedlings 13.8 min, respectively) in comparison with the standard were maintained in growth chamber at 22±1 °C under 16/8 curves. photoperiod. Some of the seedlings exposed to chrome application were taken from the growth medium on the 15th 2.5 Statistical analysis day and the remaining on the 30 th day. Shoots (without root) The experiments were carried out least 3 times using a were weighed and placed into plastic test tubes. The samples completely randomized design. The data were statistically were then frozen in liquid nitrogen and stored at -80 º C until analysed by using statistical package SPSS version 20.0 in further assay. All experiments were repeated at least three. which data subjected to ANOVA. The means were compared using Tukey’s Multiple Range Test (P≤0.05) and reported as 2.3 Extraction ±Standart Error. Plant samples were lyophilized and then crushed to powder. About 100 mg of the samples were extracted with 4 ml of 3. Results methanol for 30 minutes by a direct sonicator. The mixture 15 days of chromium application increased the hyperoside was centrifuged at 3000 rpm for 10 minutes and then the concentration in the medium containing 0.01 mM chromium supernatant transferred to a 10-ml volumetric flask. The compared to the control, whereas it decreased in the medium remaining pellet was extracted once more with 4 ml of containing 0.1 mM chromium. At 0.01 mM chrome methanol as described above. The supernatant was combined concentration, the amount of hyperoside increased 1.7-fold with the previous supernatant in a 10 ml volume borosilicate compared to the control. When the application period was amber glass tube. Then the extracts were concentrated in a increased to 30 days, the hyperoside concentration showed a vacuum centrifuge. Concentrated extracts were finalized in 4 slight increase in the seedlings on the medium containing 0.01 mL final volume with methanol and 1.5 mL samples were mM of chrome compared to the control, whereas it decreased filtered through PTFE syringe filters into HPLC vials before on the medium containing 0.1 mM of chrome (Table 1). being injected into the HPLC system. The entire extraction procedure was carried out in a day-light protected Table 1: The changes in Hyperoside amount depending on the environment. chrome concentrations and duration Chrome concentrations Exposure time 2.4 HPLC analysis of secondary metabolites (mM) 15 days 30 days HPLC analysis to detect amounts of pseudohypericin, 0(Control) 102.8±0.650d 128±0.577c hypericin, hyperoside and isoquercitrin was carried out in 0.01 176.3±0.577a 139.9±0.642b [21] accordance with the method described by Ganzer et al. 0.1 88.4±0.793e 53.3±0.750f [18] (with modifications, Yamaner et al. 2013) . In the course of The means with different letter(s) are significantly different at the our experiments, RP-HPLC DAD analysis was performed on 0.05 probability level using Tukey’s multiple range test, ±Standart an Agilent-1100 HPLC system having a photodiode array Error detector (G1315B), a degasser, a quaternary pump (G1354A), an auto-sampler (G1313), and a column oven (G1316A). A 15 days of chromium application increased the isoquercitrin Synergi MAX-RP 80 A column (150 × 4.6 mm, 4-μm particle concentration in the medium containing 0.01 mM chromium size) from Phenomenex was used for all separations. Two compared to the control, whereas it decreased up to 50% in mobile phases were used: mobile phase A: 10 mM ammonium the medium containing 0.1 mM chromium. At 0.01 mM acetate buffer (pH 5; 10 mM) and Mobile phase B: acetonitrile chrome concentration, the amount of isoquercitrin increased and methanol (9: 1). Gradient elution was carried out using the 1.8-fold compared to the control. When the application period following solvent gradient: from 87A/13B in 10 min to was 30 days, the isoquercitrin concentration did not change in 83A/17B and then in 25 min to 100 B; each run was followed the concentration of 0.01 mM chrome, whereas it decreased by an equilibration period of 10 min.

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