Mineral content and antioxidant capacity of aqueous and methanolic extract from Epilobium Hirsutum




Дата канвертавання19.04.2016
Памер48.9 Kb.

L. Vargias, et al. Scientifical Researches. Agroalimentary Processes and

Technologies, Volume XI, No. 1 (2005), 199-204

Mineral content and antioxidant capacity of aqueous and methanolic extract from Epilobium Hirsutum
L. Vargias, Monica Harmanescu, I. Gergen

Banat’s University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technology, Calea Aradului nr. 119, Timişoara, RO-300645, România, Email: igergen@yahoo.com



Abstract



In present work were investigated the total antioxidant capacity (TAC), total phenols and minerals content by different extraction conditions of Epilobium plant. The best extraction for TAC and phenols were made using methanolic solution 66% and for minerals deionized water. Aqueous extract of Epilobium, sage and peppermint were the strongest TAC in FRAP assays among the plants screened. The amount of total phenolic compounds in investigated plant extracts in most cases correlated with their antioxidant activity.
Key words: minerals, antioxidant activity, phenols content, Epilobium Hirsutum
Introduction
A great number of aromatic, spicy, medicinal and other plants contain chemical compounds exhibiting antioxidant properties. Numerous studies were carried out on some of these plants, e.g. rosemary, sage, oregano, which resulted in a development of natural antioxidant formulation for food, cosmetic and other application. However, scientific information on antioxidant properties of various plants, particularly those that are less widely used in culinary and medicine, is still rather scarce. Therefore, the assessment of such properties remains an interesting and useful task, particularly for finding new sources for natural antioxidants, functional foods and neutraceuticals.

Chemical and biological diversity of aromatic and medicinal plants depending on such factors, as cultivation area, climatic conditions, vegetation phase, genetic modifications and others is an important impetus to study flora present in different growing sites, countries and geographical zones.

This work is aimed at a preliminary screening of minerals content and antioxidant activities of the aqueous and hydroalcoholic extracts from Epilobium Hirsutum growing in Romania. To our knowledge there is a few data on minerals content antioxidant property of these plant.

The antioxidant activity of the selected plant was poorly investigated; therefor testing of their antiradical properties is of interest, primarily in order to find new promising sources for natural antioxidants, functional foods and neutraceuticals.

Free radicals can be generated by metabolic pathways within body tissues; also they can be introduced by external sources, with foods, drugs, can be caused by environmental pollution, etc. Use of natural antioxidants, as food additives for inactivated free radicals receives a lot of attention nowadays, not only for their scavenging properties, but also because they are natural, non-synthetic and their appreciation by consumers is very favorable (Miliauskas, Venskutonis & van Beek, 2004).

Oxidative stress can be reduced with the provision of additional antioxidants. Antioxidants are closely related with the prevention of degenerative illness, such as cardiovascular, neurological diseases, cancer and oxidative stress dysfunction (Bolck 1992, Diplock 1995).



Several methods are know to measure the total antioxidant capacity (TAC) of biological samples, but we tried the FRAP assay, which depends upon the reduction of ferric tripyridyltriazine complex to the ferrous tripyridyltriazine by a reductant at low pH. This ferrous tripyridyltriazine complex has an intensive blue color and can be monitored at 593 nm (Benzie & Strain, 1996). This method was elaborated for human plasma but many authors used these method for aqueous or hydroalcoholic extracts of medicinal plants (Szőllősi & Szőllősi Varga, 2002), fruits (Gil, Tomas-Barberan, Hess-Pierce & Kader, 2002) and fruit nectars (Tosun & Ustun, 2003).
Experimental
Plant material, reagents and equipment: Air-dried aerial plant material was purchased from Plafar-Romania. All chemicals and reagents were analytical grade or purest quality purchased from Sigma, Merck, Aldrich and Fluka; deionized water was used. Absorption determination for FRAP and total phenol content was made using SmartSpec spectrophotometer by Bio-Rad; Na, K, was determined using FLAPHO flamphotometer by Carl Zeiss-Jena in air-butane flame; Ca and Mg by flame absorption spectrometry using AAS1-equipment by Carl-Zeiss, Jena in air-acetylene flame.

Extraction: Dried plants were milled in a knife mill (Grindmatic-Hong Kong) and extracted with four solvents: water, methanol 33% (v/v), methanol 66% (v/v), methanol 99,5% (v/v). Extraction was applied by shaking flasks with 2-10 g (0.01 g) of plant and 100 ml of solvent in a shaking machine for 15min. The flasks were boiled on water-bath for 5 min. After cooling the samples were filtered.

Evaluation of total antioxidant capacity (TAC) (adaptation of FRAP method). Reagents: acetate buffer, 300mM/L, pH 3.6 (3.1g sodium acetate 3H2O and 16 mL conc. Acetic acid per 1L of buffer solution); 10mM/L TPTZ (2,4,6-tripyridyl-s-triazine) in 40 mM/L HCl; 20mM/L FeCl36H2O in distilled water. FRAP working solution: 25mL acetate buffer, 2.5mL TPTZ solution and 2.5 mL FeCl3 solution. The working solution must be always freshly prepared. Aqueous solution of known Fe(II) concentration was used for calibration, in a range of 0.1-1.0 mM/L. For the preparation of calibration curve 1ml aliquot of 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 M/mL aqueous Fe(II) as Mohr salts solution were mixed with 5 ml FRAP working solution; FRAP reagent was used as blank. The absorption was read after 10 min. at 25 °C at 593 nm, 1cm lights path, and the calibration curve was drawn. One mL methanolic plant extract (1g/50mL) was mixed with the same reagents as described above, and after 10 min. the absorption was measured. All determinations were performed in triplicate. Total antioxidant capacity in plant methanol extracts in Fe(II) equivalents was calculated.

The amount of total phenolic compounds: The following reagents were used: 2.0M Folin-Ciocalteu phenol reagent, gallic acid and anhydrous carbonate. The content of total phenolic compounds in plant methanolic extracts was determined by Folin-Ciocalteu method (1927). For the preparation of calibration curve 1ml aliquot of 0.16, 0.32, 0.60, 1.20, 2.0, 2.8 M/mL aqueous gallic acid solution were mixed with 10 ml Folin-Ciocalteu reagent (diluted ten-fold) and 9 mL (7.5%) sodium carbonate. The absorption was read after 2 h at 20 °C at 750 nm and the calibration curve was drawn. One mL methanolic plant extract (1g/30mL) was mixed with the same reagents as described above, and after 2 h the absorption was measured for the determination of plant phenolics. All determinations were performed in triplicate. Total content of phenolic compounds in plant methanol extracts in gallic acid equivalents (GAE) was calculated.

Mineral content: Na+ and K+, were determined using FLAPHO flamphotometer by Carl Zeiss-Jena in air-butane flame; Ca2+ and Mg2+ by flame absorption spectrometry using AAS1-equipment by Carl-Zeiss, Jena in air-acetylene flame. Na+ and Ca2+ were determinate direct in extract, K+ after 1/25 dilution and Mg2+ after 1/20, dilution. Correlation coefficient (r2) for standard curve in the linear range was 0.995 for Na+, 0.991 for K+, 0.998 for Mg2+ and 0.925 for Ca2+.
Results and Discussions
Extraction of active substances and minerals is strongly influenced by nature and concentration of solvents. For polar compound such phenols the best extraction is possible with hydroalcoholic solvents. Total antioxidant capacity and total phenols for aqueous and hydromethanolic extracts are presented in Table 1. The best extraction, with maximum antioxidant capacity and phenol content, was obtained with 66% methanol. For minerals, the best solvent is water (Table 2). To raising the methanol concentration, the mineral content for all investigated cations decreased.

Another factor, which can influence extraction, is the ratio: matter/solvent. Total antioxidant capacity for aqueous and hydro-methanolic extracts with different matter/solvent ratio is presented in Table 2. The best extraction, with maximum antioxidant capacity and phenol content, was obtained with 10/100: matter/ ethanol ratio.



Table 1. Total atioxidant capacity (as FRAP) and total phenols content by different matter/solvent ratio of Epilobium sp.

g matter/mL solvent

FRAP

M Fe(II)/mL



Total phenols

M/mL


2g matter/100 mL water

9.00.8

4.60.5

6g matter/100 mL methanol 66%

26.52.9

7.20.6

10g matter/100 mL 66 % ethanol

45.54.0

8.70.8

The better solvent for high TAC extraction is methanolic solution 66%. Also the aqueous extraction is high in TAC, but the stability of aqueous solution is poor. The better stability we found in methanolic or ethanolic solution 66%.

Comparative data for TAC and phenol content of another medicinal plant in the same extraction conditions (boiling water) are presented in Table 3 and for mineral in Table 4. From these data results that Epilobium plant presents the highest antioxidant power and phenol content.



Table 2. Total antioxidant capacity (as FRAP) and mineral content for different concentration of methanolic solutions of Epilobium sp.

Solvents

FRAP

MFe(II)/mL



Na+

g/mL


K+

g/mL


Mg2+

g/mL


Ca2+

g/mL


Water

9.00.8

1.400.11

130.012

21.02




Methanol 33 %

11.51.9

1.300.11

115.110

19.52




Methanol 66 %

15.52.0

0.950.10

71.38

13.51




Methanol 99%

7.71.1

0.550.05

30.53

8.40.9




Table 3. Total antioxidant capacity (as FRAP) and total phenols content by different medicinal plant species (Extraction: 2g matter/100 boiled water)

Medicinal plants

FRAP

M Fe(II)/mL



Total phenols

M/mL

mg GAE/g plant

Epilobium sp.

9.00.8

4.60.5

39.14.2

Hops (Strobuli lupus)

5.00.4

1.80.2

15.31.7

Peppermint (Herba Menthae)

7.80.8

4.00.3

34.02.5

Sage (Folium Salviae)

5.10.5

1.60.2

13.61.6

Chamomile (Matricaria Chamomilla)

1.20.1

0.20.01

1.70.09

Linden (FloresTiliae)

0.20.01

0.010.001

0.080.009

Table 4. Mineral content of aqueous extract of different medicinal plants (Extraction: 2g matter/100 boiled water)

Medicinal plants

Na+

g/mL


K+

g/mL


Mg2+

g/mL


Epilobium sp.

1.40.1

130.012

21.02

Hops (Strobuli lupus)

1.00.1

255.026

28.03

Peppermint (Herba Menthae)

17.20.2

255.027

76.07

Sage (Folium Salviae)

1.40.1

160.015

36.05

Chamomile (Matricaria Chamomilla)

3.60.4

142.015

10.01

Linden (FloresTiliae)

0.40.06

30.04

1.20.1

Higher content was identified for potassium, followed by magnesium and sodium. Only in peppermint sodium is in high quantity, possible that this plant is fond in salinized soil (Table 4). High mineral content was identified in peppermint, hops and sage.

In methanolic extract, Miliauskas, Venskutonis & van Beek (2004) find that sage extract is highest in TAC and total phenol that matricaria extract. The same result is evident also from our data. The total phenol compound identified by they in methanolic extract was 23 mg/g plant in sage and 7.5 mg/g plant in chamomile. Epilobium extract is higher in TAC and phenols than sage in water extract. That mind, the Epilobium plant are promising plant for antioxidant compounds extraction.



Conclusions
Aqueous extract of Epilobium, sage and peppermint were the strongest TAC in FRAP assays among the plants screened. Epilobium plant is promising plant for more detailed investigation of their antioxidant properties and application possibilities. Their antioxidant power was higher than that sage (Salviae officinalis) which is one of the most effective plants in terms of antioxidant activity. The amount of total phenolic compounds in investigated plant extracts in most cases correlated with their antioxidant activity. High mineral content, especially K, was identified in peppermint, hops and sage.
References
Benzie I.F.F. and Strain J.J. (1996). Ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: The FRAP assay, Anal. Biochemistry, 239, 70-76.

Bolck G. (1992). A role of antioxidants in reducing cancer risks, Nutritional Revue, 50, 207-213.

Diplock A. (1995). Antioxidant nutrients-efficacy in disease prevention and safety, Biochemist, 17, 16-18.

Folin O. and Ciocalteu V. (1927). On tyrosine and tryptophane determination in proteins, Journal of Biological Chemistry, 27, 627-650

Miliauskas G., Venskutonis P.R. and T.A. van Beek. (2004). Screening of radical scavenging activity of some medicinal and aromatic plants extracts, Food Chemistry, 85, 231-237.

Szőllősi R. and Szőllősi-Varga I. (2002). Total antioxidant power in some species of Labiatae, Acta Biologica Szegediensis, 46, 125-127

Tosun I. and Ustun N.S. (2003). An investigation about antioxidant capacity of fruit nectars, Pakistan Journal of Nutrition , 2, 167-169.

Gil M.I., Tomas-Barberan F.A., Hess-Pierce B. and Kader A.A. (2002). Atioxidant capacities, phenolic compounds, carotenoids and vitamin C content of nectarine, peach and plum cultivars from California, J. Agric. Food Chem., 50, 4976-4982







База данных защищена авторским правом ©shkola.of.by 2016
звярнуцца да адміністрацыі

    Галоўная старонка