Pollination Effects on Antioxidant Content of Perilla Frutescens Seeds Analysed by NMR Spectroscopy

Natural Product Research Formerly Natural Product Letters

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Pollination effects on antioxidant content of Perilla frutescens seeds analysed by NMR spectroscopy

Paola Ferrazzi, Monica Vercelli, Amina Chakir, Abderrahmane Romane, Monica Mattana & Roberto Consonni

To cite this article: Paola Ferrazzi, Monica Vercelli, Amina Chakir, Abderrahmane Romane, Monica Mattana & Roberto Consonni (2017) Pollination effects on antioxidant content of Perilla frutescens seeds analysed by NMR spectroscopy, Natural Product Research, 31:23, 2705-2711, DOI: 10.1080/14786419.2017.1292267 To link to this article: https://doi.org/10.1080/14786419.2017.1292267

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Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=gnpl20 Natural Product Research, 2017 VOL. 31, NO. 23, 2705–2711 https://doi.org/10.1080/14786419.2017.1292267

Pollination effects on antioxidant content of Perilla frutescens seeds analysed by NMR spectroscopy

Paola Ferrazzia, Monica Vercellia, Amina Chakirb, Abderrahmane Romaneb, Monica Mattanac and Roberto Consonnid aDepartment of Agricultural, Forestry and Food Science, University of Turin, Grugliasco, Italy; bFaculty of Sciences Semlalia, Department of Applied Organic Chemistry, University Cadi Ayyad, Marrakech, Morocco; cInstitute of agricultural biology and biotechnology (IBBA), Milano, Italy; dInstitute for macromolecular studies (ISMAC), Milano, Italy

ABSTRACT ARTICLE HISTORY The effects ofPerilla frutescens pollination on the content of seed Received 17 October 2016 antioxidants were analysed by agronomical and pollination trials, Accepted 30 January 2017 comparing seeds produced from bagged plants in 2013 (A) to KEYWORDS prevent access to pollinating insects, and seeds from open-pollinated Perilla frutescens; pollination plants in 2013 (B) and 2015 (C). The seeds of open-pollinated plants trials; NMR; rosmarinic acid; were significantly more numerous and heavier than those of self- flavonoids; antioxidant pollinated plants. 1H NMR seed analysis showed a higher presence implementation; honey bee of phenolic compounds in open-pollinated seeds, mainly rosmarinic pollination effects acid and flavonoids, apigenin and luteolin. Flavonoids were present in the glucosylated form in seeds (A) and (C), and in the aglycone form in seeds from (B) plants. Saturated and unsaturated fatty acids (palmitic, linoleic and linolenic) were more abundant in seeds from self-pollinated flowers. Pollination performed almost exclusively by the honeybee notably increased the antioxidant content in perilla seeds and gave rise to a reduction in the fatty acid content.

CONTACT Monica Vercelli [email protected] Presented to CIPAM 2016, 6th International Congress of Aromatic and Medicinal Plants, 29 May–1 June 2016, Coimbra (Portugal). supplemental data for this article can be accessed at http://dx.doi.org/10.1080/14786419.2017.1292267. © 2017 Informa UK Limited, trading as Taylor & Francis Group 2706 P. FERRAZZI ET AL.

1. Introduction Perilla frutescens (L.) Britton, family Lamiaceae, Shiso or Egoma, a species native to eastern Asia where it is commonly consumed as a vegetable, is increasingly known for its medicinal properties and high antioxidant content (Sargi et al. 2013; Bachheti et al. 2014). The interest in this plant is also related to its anticarcinogenic (Bardon et al. 2002; Asif 2011), anti-inflammatory (Osakabe et al. 2002; Ueda et al. 2002), and antiallergic action (Takano et al. 2004; Igarashi & Miyazaki 2013), as well as the cosmetic properties derived from seeds and oil (Murai et al. 2001) and as a source of compounds for mosquito management (Tabanca et al. 2015). Moreover, P. frutescens has been demonstrated, in Italy, to be an interesting plant for honeybees (Consonni et al. 2013), which collect mainly nectar during the flowering period in September (Barbieri & Ferrazzi 2011). This plant provides important sources of food in a period of bloom shortage, useful for supplying colonies in winter. NMR analyses performed in Italy on Perilla flowers and leaves showed their richness in antioxidant compounds, above all linolenic, and rosmarinic acids (Consonni et al. 2013). The positive effects of pollinating insects, and in particular the honeybee, are well known in crop plants such as fruits, oilseeds and fodder (McGregor 1976; Klein et al. 2007; Jauker et al. 2012; Klatt et al. 2014; Marini et al. 2015), and as a nutrient supplement to animals (Peiretti et al. 2010, 2011), but few studies have been carried out on medicinal plants (Salami et al. 2016). In medicinal plants, the biosynthesis of bioactive components is strongly influenced by a number of environmental factors (Taarit et al. 2009) as well as by maturity stage (Peiretti 2011), morphological structure and pollination system (Free 1993; Delaplane & Mayer 2000). The seeds from P. frutescens are the most important part of the plant, and pollination can greatly affect their production and chemical composition. The present study explored the possible effects of pollinator action on seed production and content of active ingredients in seeds from this species analysed by NMR. The actual and serious decline of pollinators, both of the honeybee and wild bees (Lebuhn et al. 2013), may also have negative effects on the seed yield of Perilla and content of valuable metabolites.

2. Results and discussion 2.1. Self- and outcrossing pollination The main pollinator of P. frutescens is the honeybee, according to previous studies on Perilla- visiting insects (Barbieri & Ferrazzi 2011; Consonni et al. 2013). Occasionally, other Apoidea, Syrphid flies and Lepidoptera Rhopalocera are also registered. The seeds of P. frutescens plants enclosed in net cages to exclude pollinators in 2013 (A) and seeds of Perilla plants permitted to outcrossing in 2013 (B), and 2015 (C) were examined (Table 1). Seed number values from 2013 showed that open-pollinated plants (B) produced a higher number of seeds than self-pollinated plants (A). Statistical significances were highlighted. Regarding seed weight, significant differences were observed in B compared with A. The highest values per plant were registered in 2015 open-pollinated plants (C) (Table 1). The 1000-seed weight from A, B and C is indicated in Table 1. NATURAL PRODUCT RESEARCH 2707

The larger number and greater weight of seeds from open-pollinated plants highlighted the beneficial effects of open-pollination compared with self-pollination, as described by Bommarco et al. (2012) on oilseed rape.

2.2. NMR-based metabolite identification from Perilla seeds Alcoholic extracts of seeds from self-pollinated in 2013 (A) and open-pollinated flowers collected in both 2013 (B) and 2015 (C) by non-caged plants were submitted to NMR inves- tigations to evaluate differences in metabolite content. The aliphatic region of1 H NMR spectra showed mainly the presence of saccharides (α, β glucose and sucrose), saturated (SFA) and unsaturated (UFA) fatty acids (palmitic and/or stearic, linolenic and linoleic, respectively) (Figure S1), according to Shin and Kim (1994), while the aromatic region revealed the presence of large amount of rosmarinic 3-glucoside (R3 g), luteolin 7-glucoside (L7 g) and apigenin 7-glucoside (A7 g), together with the corresponding aglycone forms (R, L, A) (Figure S2), whose presence is most likely due to the activity of glycosidic enzymes. Concerning fatty acid content, they were present in larger amounts in seeds from (A) compared to (C) (more than 60%) and (B) (about 30%). Regarding linolenic acid, seeds from (A) had about 25% more than seeds from (B) and (C). Conversely, linoleic acid content was found in greater amounts in seeds from (C) than the other two types of seeds. Finally, the sucrose content was relatively similar in all types of seeds (Figure 1).

Table 1. Pollination system effects on Perilla seed number and weight (mean values) and 1000-seed weight.

Pollination systems Seed/plant Seed weight/plant 1000 seed weight (g) Self-pollinated plants (A) 2013 124.8** (A vs. B) 0.05** (A vs. B) 0.40 Open-pollinated plants (B) 2013 337.6ns (B vs. C) 0.32ns (B vs. C) 0.95 Open-pollinated plants (C) 2015 301.4 0.39 1.23 Notes: ns, and **, non significant or significant at P ≤ 0.01, respectively.

Figure 1. Quantification of selected aliphatic metabolites in Perilla seed extracts referred to the total metabolite content of the extract. 2708 P. FERRAZZI ET AL.

Figure 2. Quantification of selected aromatic metabolites in Perilla seed extracts referred to the total metabolite content of the extract.

By comparison of seed extracts from 2015 open-pollinated plants (C) and self-pollinated plants (A), the content of L7 g, A7 g, R3 g and R in (C) was about three times greater than seeds (A). Interestingly, L and A were present only in seeds from (B). R3 g was always present in higher amounts than R content for all types of seeds. In particular, in (C) R3 g was about two times greater compared to (A), while the R content was more than three times higher. Seeds from (B) had slightly less (about 15%) R3 g and R than seeds (C) (Figure 2). Rosmarinic acid and glucosylated rosmarinic acid, luteolin and apigenin were detected in Italy as major phenolic components in Perilla seeds, according to Ha et al. (2012), Lee et al. (2013) and Zhou et al. (2014), although caffeic acid reported by the previous authors was not detectable in the present study. Rosmarinic acid and glycosylated rosmarinic acid were found in all seeds, with percentages notably higher in open-pollinated plants. The presence of the flavonoid apigenin and luteolin in the glycosylated form in seeds from self-pollinated (A) and 2015 open-pollinated plants (C), which were not detected in 2013 open-pollinated plants (B), suggests that ageing is related to de-glycosylation processes, which are absent in fresh seeds and probably not triggered by the lack of cross-pollination. However, the quantity of flavonoids was significantly higher in open-pollinated plants (B and C). Otherwise, self-pollinated seeds had a higher content of fatty acids, even if their weight was lower than those in open-pollination. This is in line with observations in several other plant species, where poorly pollinated plants invest more into fewer seeds using self- or wind pollination when insect pollination is lacking (Jauker et al. 2012).

3. Experimental 3.1. Plant material Seeds from P. frutescens (L.) Britton var. crispa (Bentham) Deane ex Bayley forma purpurea Makino from former crops were sown in April 2013 and 2015 in rows at Bussoleno, Turin province, northwestern Italy, 470 m a.s.l. NATURAL PRODUCT RESEARCH 2709

Voucher specimens were deposited at the Herbarium of the Department of Life Sciences and Systems Biology at the University of Turin (viale Mattioli 25, Turin, Italy; voucher No. 2654, TO-HG section Herbarium generale).

3.2. Agronomical and pollination experiments In this study, pollination tests were conducted in September 2013 on P. frutescens by isolating plants and considering plants left open to insect pollination. Six plants of Perilla were bagged before blooming with cages incorporated with 1 mm nets to prevent access to pollinator visits (A), and six plants were left open to allow outcrossing (B). The experiment was conducted in duplicate. Seeds were harvested at full maturity stage when they were completely dried, and kept at room temperature. In September 2015, additional trials were carried out to assess possible differences in antioxidant content of six open-pollinated plants (C), in duplicate; all seeds were counted and weighed. The weight of 1000 seeds was quantified. Periodic surveys (every three days) were carried out during the period of Perilla flowering on non-caged plants to detect insect pollinators; the products harvested were monitored with regard to honeybee nectar and/or pollen foraging activity, according to Barbieri and Ferrazzi (2011).

3.3. Extraction and isolation 150 mg of P. frutescens seeds from (A), (B), (C) were ground in a mortar under liquid nitrogen and extracted with 1.4 mL of deuterated methanol (CD3OD 80%). The solution was vortexed for 5 min and stirred overnight. The solution was then centrifuged for 20 min at 12,000 g, and 600 μL of the supernatant was used for NMR analysis.

3.4. General experimental procedures Spectra were recorded at 300 K with a 600 MHz Bruker DRX spectrometer (Bruker Biospin GmbH Rheinstetten, Karlsruhe, Germany) operating at 14.1 T and equipped with 5 mm TXI probe with z gradient. Monodimensional 1H NMR spectra were recorded with standard pulse sequences incorporating a solvent suppression scheme based on low-power solvent irradiation. Bidimensional homo- and hetero-nuclear correlated spectra (TOCSY and HSQC, HMBC) were also acquired for the resonance assignment of metabolite content. All spectra were recorded and processed using TOPSPIN software (v. 1.3 and 3.5 Bruker Biospin). Resonance assignment was performed with the aid of ‘homemade’ and public databases (BRMB, and HMDB, Wishart et al. 2013) of metabolites, already published data, and in addition, L, L7 g, A and A7 g have been purchased from Sigma Aldrich Co, St Luis, M.O., U.S.A., purity >97%). Alcoholic extracts have been performed on mixtures of seeds collected from several plants, in order to minimise possible metabolic differences among similar plants of the same batch. NMR signals of selected metabolites have been quantified respect to the total amount of metabolites.

3.5. Statistical analysis Seed number and weight values were subjected to Independent Samples T-test using the SPSS statistical package (v. 22.0, SPSS Inc., Chicago, USA). 2710 P. FERRAZZI ET AL.

4. Conclusion According Paiva et al. (2003) and Bommarco et al. (2012), the larger number and greater weight of seeds from open-pollinated plants highlight the beneficial effects of pollination compared with the absence of bees and self-pollination. In fact, even if P. frutescens is a self-fertilising crop (Tong et al. 2015), the difference between seeds from caged and open- pollinated plants is highly significant. Considering the metabolic profile, rosmarinic acid and glycosylated rosmarinic acid, luteolin, and apigenin were the major phenolic compounds from Perilla seeds cultivated in Italy. Our data strongly support the effects of pollination in increasing the Perilla content of antioxidant compounds, such as rosmarinic acid and flavonoids, while decreasing the fatty acid content. The content of phenolic compounds, highest in seeds from open-pollinated plants, seems to stress the importance of pollination by insects evidently able to improve the production of these metabolites. The presence of Apis mellifera, the most abundant P. frutescens pollinator, and the pollinator biodiversity conservation in the environment, could be essential to ensure greater seed production and increase the content of active ingredients in this medicinal plant.

Disclosure statement No potential conflict of interest was reported by the authors.

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