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 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 . 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 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. The flow rate of the by about 3 times in the medium containing 0.1 mM chrome mobile phase was 1 mL/min. An aliquot of 50 μL of extract (Table 2). per sample was injected onto the column and detection was carried out at 270 nm wavelength for pseudohypericin and Table 2: The changes in Isoquercitrin amount depending on the hypericin and at 590 nm wavelength for hyperoside and chrome concentrations and duration isoquercitrin. All separations were carried out with a fixed column temperature of 40 °C. Comparison of the UV spectra Chrome concentrations Exposure time and retention times of standard injection peaks against sample (mM) 15 days 30 days 0(Control) 10.70±0.321c 15.40±0.230b peaks was used to assign peak identity. Standard curves were 0.01 19.70±0.650a 15.70±0173b achieved by plotting the peak areas of standard dilutions of 0.1 8.50±0.680d 5.9±0.208e hypericin (seven set of standard dilutions, between 0.02-10 The means with different letter(s) are significantly different at the μg/mL), pseudohypericin (six set of standard dilutions,

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0.05 probability level using Tukey’s multiple range test, ±Standart elicitor’s exposure time and elicitor’s concentration [23]. Error The best results were obtained in 15-day applications in media Pseudohypericin content at 0.01 mM concentration of chrome containing 0.01 mM chrome for all the secondary metabolites was 2.2 times higher than that of the control seedlings in 15 in our study. In Trillini et al. [24] experiments, an increase days of application. The increase in the amount of about 4-fold of amount of secondary metabolite by 7-days pseudohypericin in the medium containing 0.1 mM chrome is chrome application at 0.1 mM concentration was recorded. 1.3-fold of the control. In 30 days application experiment, an Rai et al. reported increasing proline and eugenol production increase for pseudohypericin content was observed in 0.01 in Ocimum tenuiflorum L. [25] and Vernay et al. reported to mM chrome concentration (1.3-fold), but not in the case of 0.1 increse proline and scopolamine production in Datura innoxia mM chrome concentration (Table 3) Mill with chrome application [26]. Effect of chromium on secondary metabolite induction varies among species. Table 3: The changes in Pseudohypericin amount depending on the Chrome, a toxic metal, causes oxidative stresses in [27- chrome concentrations and duration 29] . Oxidative stress induces enzymatic (CAT, SOD etc.) and [9] Chrome concentrations Exposure time non-enzymatic (glutathione, ascorbate, etc.) antioxidants . (mM) 15 days 30 days As known well, secondary metabolites are formed under stress 0(Control) 5.60±0.305d 7.3±0.173c conditions to support defense mechanism [30]. In the present 0.01 12.70±0.305a 9.90±0.346b study, Cr stress may produce reactive oxygen species which 0.1 7.20±0.519c 7.10±0.152c stimulates accumulation of hypericins and flavonoids in The means with different letter(s) are significantly different at the Hypericum adenotrichum Spach. However, further 0.05 probability level using Tukey’s multiple range test, ±Standart investigations are required to determine the relationship Error between chrome effect and antioxidant mechanism of H. adenotrichum Spach The amount of hypericine increased about 1.7 times compared to the control in the seedlings exposed to 0.01 mM of chrome 5. Conclusıon for 15 days. When the period of application was increased to The study we carried out for the elicitation of secondary 30 days, no significant change were observed in hypericin metabolites by applying chrome is the first for H. content compared to the control (Table 4). adenotrichum plant, and it can be a pioneer in carrying out more detailed studies on this subject. This study using ın vitro Table 4: The changes in Hypericin amount depending on the chrome seedlings has contributed to our knowledge of the behavior of concentrations and duration Hypericum species under stress conditions and our data can be Chrome concentrations Exposure time used to understand the pathways of synthesis of the secondary (mM) 15 days 30 days metabolites and the factors affecting them. It is also believed 0(Control) 5.50±0.251c 7.20±0.458bc that Hypericum adenotrichum is also useful for experimental 0.01 9.60±0.416a 7.70±0.378b botanical and agricultural technologies to be determined for 0.1 8.20±0.493ab 6.90±0.208bc use in the medical industry. The means with different letter(s) are significantly different at the 0.05 probability level using Tukey’s multiple range test, ±Standart 6. Aknowledgement Error This study was financially supported by Adnan Menderes University Scientific Research Project Department (Project 4. Dıscussıon no: FBE- 09013). Abstract part of the study was presented at It is commonly accepted that the type of culture is more the 21st National Biology Congress, 3-7 September 2012, decisive than the type of elicitor on compoundswhich are İzmir TURKEY. desired to induce in H. perforatum by elicitation. One of the elicitor induced compounds are hypericins, experimented in 7. References seedlings and shoot cultures (probably having hypericin 1. 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