ANTIOXIDANT ACTIVITY and PHENOLICS CONTENT of Capsella Bursa-Pastoris and Marrubium Vulgare DEPENDING on ENVIRONMENTAL FACTORS
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Environmental Engineering and Management Journal July 2019, Vol. 18, No. 7, 1553-1560 http://www.eemj.icpm.tuiasi.ro/; http://www.eemj.eu “Gheorghe Asachi” Technical University of Iasi, Romania ANTIOXIDANT ACTIVITY AND PHENOLICS CONTENT OF Capsella bursa-pastoris AND Marrubium vulgare DEPENDING ON ENVIRONMENTAL FACTORS Elena Neagu1, Gabriela Paun1, Oana Ungureanu 1, Gabriel Lucian Radu2 1Centre of Bioanalysis, National Institute of Research and Development for Biological Sciences, 296 Splaiul Independentei, 060031, Bucharest 6, Romania 2Faculty of Applied Chemistry and Materials Science, University Politehnica of Bucharest, Str. Gh. Polizu Nr. 1-7, 011061 Bucharest 1, Romania Abstract In this work we aimed to examine the influence of the main abiotic factors (temperature, rainfall, relative humidity, atmospheric pressure) on the synthesis of active compounds (phenolics, flavonoids), with antioxidant and therapeutic effect of two herbs - Capsella bursa-pastoris (shepherd's purse) and Marrubium vulgare (horehound) collected from two different areas of Romania: Dobrogea (Constanta) and Muntenia (Bucharest). The studies have shown that biogenesis and accumulation of secondary metabolites that gives the therapeutic properties of medicinal plants is influenced by biotic and abiotic factors. In this work we aimed to determine the profile of concentrations of biologically active compounds from the studied herbs depending on the harvest time period and variability of the environmental factors. The medicinal herbs had harvested in autumn (September, 2013) and in spring (May – June, 2014); the alcoholic and aqueous extracts were analyzed in terms of composition: in main biologically active compounds (phenolics, flavonoids) and antioxidant activity as DPPH and ABTS, and spectral data analysis by FTIR. Experimental data obtained showed a significantly higher phenolics and flavonoids content and also a higher antioxidant activity in herbs harvested from the Dobrogea (Constanta) area, for the herbs studied. Key words: abiotic factors, antioxidant activity, Capsella bursa-pastoris, flavonoids, Marrubium vulgare, phenolics Received: December, 2017; Revised final: June, 2018; Accepted: July, 2019; Published in final edited form: July, 2019 1. Introduction Environmental factors such as temperature, fertility, light intensity, the rich mineral content in the The secondary metabolites from plants are water and CO2 influence the growth of plants and sources for additives and flavors used in secondary metabolite production (Ramakrishna and pharmaceuticals and alimentary industry. These Aswathanarayana, 2011; Vincze et al., 2018). metabolites are vital in the plant's adaptation process Variations in the aforementioned elements impact the to the environment and to overcoming stress quality and properties of wild and cultivated extracts conditions and can generate free radicals. These can for additional developmental applications (Szakiel et react with certain kind of molecule: DNA, proteins al., 2011). and lipids resulting into membranes changes which Examples in reference to the influence of further determines cell and tissue's injuries (Abdi and abiotic factors on the secondary metabolic synthesis Ali, 1999). from herbs are shown in Table 1. Author to whom all correspondence should be addressed: e-mail: [email protected]; [email protected]; Phone: +402200900; Fax: +402200900 Neagu et al./Environmental Engineering and Management Journal 18 (2019), 7, 1553-1560 Table 1. The influence of the abiotic factors on the level of secondary metabolites Abiotic factors Plant species Effect References Ban et al. (2007) Lower temperature Ubi et al. (2006) Malus sp. Higher levels of anthocyanins UV-B irridiation Lin-Wang et al. (2011) Panax Long exposure to light Ginsenosides in high amount Fournier et al. (2003) quinquefolius Higher altitudes and lower Arnica montana Phenolics derivatives Albert et al. (2009) temperatures Activation of flavonoid Solar radiation exposure Vaccinium myrtillus Jaakola et al. (2004) biosynthesis Drought Quercus ilex Decrease of monoterpene synthesis Lavoir et al. (2009) Plants belonging to the same species but Muntenia and Dobrogea, in two seasons as well as occurring in different geographical zones may differ associations with some environmental factors. significantly in their content of particular secondary metabolites, both qualitatively and quantitatively 2. Materials and method (Szakiel et al., 2011). The study on the interaction between plant secondary metabolism and 2.1. Preparation of plant sample environmental factors may be helpful to the cultivation and standardization of traditional The medicinal herbs - Capsella bursa-pastoris medicinal herbs and may provide a new approach to and Marubium vulgare - were harvested in autumn increasing target secondary metabolites. (September, 2013) and in spring (May – June, 2014) Capsella bursa-pastoris (Shepherd's Purse) from two different Romania areas: Dobrogea (fam. Brassicaceae-Cruciferae) is a herbaceous plant (Constanta) and Muntenia (Bucharest). The aerial species which occurs very frequently in the parts of plants (leaves, stems and inflorescences) were spontaneous flora of Romania. The plant is known for dried at the oven to 40oC then were ground in a mill its curative qualities from antiquity, when it was used grinder (GRINDOMIX). as a laxative and aphrodisiac. We have obtained two types of extracts: In folk medicine it is used as a uterine tonic, aqueous, 8% weight with distilled water at 60ºC (8 hemostatic and healing. For medicinal purposes, grams the ground herb at 100 mL distilled water) and shepherd's purse are harvested in spring (April-May) hydro-alcoholic extracts, 8% weigh with 50% and has many therapeutic uses: tonics, stimulants, ethanolic solution (8 grams the ground herb at 100 mL hemostatic, healing, astringent, utero-tonic, coronary ethanolic solution with 50% concentration). All vasodilators, hypotensive, anti-inflammatory extracts were prepared by sonication for 1 h and then analgesic and anti-ulcer (Kuroda and Takagi, 1969). filtered through filter paper. Clear extracts obtained Marubium vulgare (Horehound) (fam. were stored at 4ºC until bioanalysis extracts was Laminaceae) is a plant of wild flora, widespread in performed. Europe. In Romania it is spread much in the steppe of Oltenia, Muntenia, Moldova and all over in Dobrogea. 2.2. Total phenolic content For therapeutic purposes use leaves, flowers and young stems, harvested between June and September. The total phenols’content determination from Many of the effects ascribed to traditional medicine of plant extracts was performed by the Folin-Ciocalteu the horehound have been confirmed in recent years of spectrophotometric method (Singleton et al., 1999). In scientific research and clinical studies, as: antioxidant brief, at 2.5 mL sample were added 2.5 mL Folin – (Berrougui et al., 2006), analgesic (Meyre-Silva et al., Ciocalteu reagent, shaken and filtered, then 1 mL of 2005), anti oedematogenic (Stulzer et al., 2006), filtrate was mixed with 9.5 mL sodium carbonate 20% antidiabetic type II (Herrera-Arellano et al., 2004) and and after 2 minutes the absorbance was read at 760 in neurological disorders very recently (Orhan et al., nm. The phenolic content in the tested samples was 2010). This species is less used in phytotherapy. There calculated based on the calibration curve obtained are not data regarding the influence of abiotic factors between 0-100 mg/L gallic acid (GAE), with R2 on the phenolics and flavonoids amount of Capsella =0.9969. bursa-pastoris and Marubium vulgare. Since the cellular antioxidative status plays an 2.3. Total flavonoid content important role in the capacity of plants to cope with oxidative stress induced by environmental factors, the The total flavonoid content was determined aim of the current work was to compare the plant according Lin method (aluminum chloride based secondary metabolites (phenolics and flavonoids) method), with slight modifications (Lin and Tang, production and the antioxidant activity from the same 2007). Briefly, at 5 mL of the extract were added 7.5 species harvested from different geographical regions: mL of MeOH and filtered. At which point, 1 mL of the 1554 Antioxidant activity and phenolics content of Capsella bursa-pastoris and Marrubium vulgare depending on environmental factors resultant sample was transferred to a 5 mL bottle, then http://rp5.ru and http://freemeteo.com. Results are 1 mL of 10% sodium acetate solution, 0.6 mL of 2.5% presented as means for each month in the period from aluminum chloride hexahydrate solution and 0.5 mL January to December. MeOH were added. After 15 min, the absorbance of the mixture was metered at 430 nm and the flavonoid 2.7. Statistical analysis content expressed in mg rutin equivalent (RE)/L was measured using the calibration curve in the 0-120 The Statistical Analysis was done using test mg/L concentration range and R2 = 0.991. Microsoft Office Excel 2007. 2.4. Evaluation of the antioxidant activity of the 3. Results and discussions extract was carried out spectrophotometrically using two comparative modes: 3.1. Determination of total phenolics and flavonoids contents 2.4.1. DPPH assay Briefly 100 µL extract were mixed with 1 mL The results regarding bioactive compounds DPPH (2,2-diphenyl-1-picrylhydrazyl) solution (0.25 (phenolics, flavonoids) of vegetal extracs were mM) and 1.9 mL methanol. The absorbency was presented in Fig. 1 and Fig. 2. measured at 517 nm, after 3 min with slight modifications